Methods and compositions for determining the purity of and purifying chemically synthesized nucleic acids

ABSTRACT

An antibody microarray is described comprising a plurality of antibodies immobilized on a substrate, wherein each antibody specifically binds to a synthetic oligomer (e.g., an oligonucleotide or oligopeptide) having an organic protecting group covalently bound thereto, which antibody does not bind to that synthetic oligomer when the organic protecting group is not covalently bound thereto. Methods of making and using such antibodies are disclosed, along with cells for making such antibodies. Methods of making and using such antibody microarrays are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of International ApplicationNo. PCT/US02/20418, filed Jun. 28, 2002, which designated the UnitedStates and was published in English on Jan. 9, 2003 as InternationalPublication No. WO 03/003014, and which claims the benefit of U.S.Provisional Application No. 60/302,153, filed Jun. 29, 2001. The entiredisclosures of International Application No. PCT/US02/20418 and U.S.Provisional Application No. 60/302,153 are hereby incorporated herein byreference.

FIELD OF THE INVENTION

[0002] The present invention relates to the detection, identification,and quantification of protecting groups remaining after chemicalsynthesis of oligomers, particularly oligonucleotides, and also relatesto the purification of such oligomers.

BACKGROUND OF THE INVENTION

[0003] Over the past decade automated chemical synthesis of nucleicacids such as DNA and RNA on solid supports has been developed. Thesechemical processes include the use of agents to protect the exocyclicamines of the nucleotide bases adenine, thymine, cytosine and guanineand to direct the synthesis by blocking the 2′OH of RNA's ribose. Thebases within the nucleic acid product of the synthesis are deprotectedupon cleavage of the nucleic acid from the solid support. However, theextent of base deprotection is not easily determined.

[0004] For example, after base deprotection of synthetic RNA, productsstill contain the 2′-dimethylsilyl tert-butyl group as a protection ofthe 2′OH of the ribose moiety. This protecting group is removedcarefully by chemical means so as not to effect the chemistry andstructure of the RNA. However, the extent of deprotection of the 2′OH isnot readily determined. The nucleic acid is purified by high pressureliquid chromatography or by gel electrophoresis. However, some of theunwanted products of the synthesis are complete nucleic acid sequencesthat still contain one or more protecting groups, and shorter than fulllength (aborted) sequences difficult to separate from full lengthsequences, especially for oligomers of longer than 50 nucleosides. Atpresent, there is no easy method to determine how much of eachprotecting group, if any, still remains on the product, and whatproportion of the product is full-length. See generally Davis, G. E.,Gehrke, C. W., Kuo, K. C., and Agris, P. F. (1979) Major and ModifiedNucleosides in tRNA Hydrolysates by High Performance LiquidChromatography. J. Chromatogr. 173:281-298; Agris, P. F., Tompson, J.G., Gehrke, C. W., Kuo, K. C., and Rice, R. H. (1980) High-PerformanceLiquid Chromatography and Mass Spectrometry of Transfer RNA Bases forIsotopic Abundance. J Chromatogr. 194:205-212; Gehrke, C. W., Kuo, K.C., McCune, R. A., Gerhardt, K. O., and Agris, P. F. (1981) QuantitativeEnzymatic Hydrolysis of tRNAs: RP-HPLC of tRNA Nucleosides. J.Chromatogr. 230:297-308; Chromatograghy and Modification of NucleosidesVolumes A, B and C (Gehrke, C. W. and Kuo, K. C. T., eds.), ElsevierPublishing Co. 1990; Agris, P. F. and Sierzputowska-Gracz, H. (1990)Three Dimensional Dynamic Structure of tRNA's by Nuclear MagneticResonance. In Chromatography and Modification of Nucleosides (Gehrke, C.W. and Kuo, K. C. T., eds.), Elsevier Publishing Co., pp. 225-253;Agris, P. F., Hayden, J., Sierzputowska-Gracz, H., Ditson, S., Degres,J. A., Tempesta, M., Kuo, K. C. and Gehrke, C. W. (1990) Compendium onBiological, Biochemical, Chemical, Physical and Spectroscopic Propertiesof RNA and DNA Nucleosides. In Chromatography and Modification ofNucleosides, Elsevier Publishing Co.

[0005] The incomplete removal of the protecting group and lack of asimple assay is a problem for two industries and for numerousresearchers world wide: (i) the multitude of companies now providingnucleic acid sequence synthesis products by overnight delivery havedifficulty telling their customers the extent to which the product isdeprotected; (ii) pharmaceutical companies cannot easily verify forregulatory agencies the purity and/or length of the therapeutic ordiagnostic oligonucleotide products they seek to introduce or market.Accordingly, there is a need for simple and reliable techniques fordetermining the purity of oligonucleotide products and for determiningthe proportion of full length oligonucleotide products.

SUMMARY OF THE INVENTION

[0006] The present invention relates to an antibody microarray thatcomprises a plurality of antibodies immobilized on a substrate and theuse thereof. Each of the antibodies immobilized on the antibodymicroarray specifically binds to a synthetic oligomer having an organicprotecting group covalently bound thereto, which antibody does not bindto the synthetic oligonucleotide when the organic protecting group isnot covalently bound thereto.

[0007] An antibody chip according to the present invention may be usedto detect incomplete deprotection of synthetic oligomers and/or separateprotected synthetic oligomers from fully deprotected syntheticoligomers. Such an antibody chip may also be used to detect incompleteelongation of synthetic oligomers and/or separate full-length oligomersfrom incomplete synthetic oligomers. The methods described herein may beuseful in high throughput assays of unbound oligomers or oligomers boundto a substrate such as an oligonucleotide microarray. For example, anantibody chip according to the present invention and methods of usingsuch an antibody chip may be useful in a high throughput assay fordetecting the insufficient deprotection or elongation of DNA used toform a DNA chip.

[0008] One aspect of the invention includes a method of detectingincomplete deprotection of a synthetic test oligonucleotide using anantibody microarray. The method comprises the following steps:

[0009] (a) providing an antibody microarray comprising a plurality ofantibodies immobilized on a substrate, each antibody specificallybinding to a synthetic oligonucleotide having an organic protectinggroup covalently bound thereto, which antibody does not bind to thesynthetic oligonucleotide when the organic protecting group is notcovalently bound thereto;

[0010] (b) contacting the synthetic test oligonucleotide with theantibodies immobilized on the antibody microarray; and

[0011] (c) detecting binding of the synthetic test oligonucleotide tothe antibodies immobilized on the antibody microarray, the presence ofbinding indicating incomplete deprotection of the synthetic testoligonucleotide.

[0012] Another aspect of the invention includes a method of purifying asynthetic oligonucleotide mixture prepared using organic protectinggroups. The method uses an antibody microarray and comprises thefollowing steps:

[0013] (a) providing a synthetic oligonucleotide mixture comprisingfully deprotected synthetic oligonucleotides, partially deprotectedsynthetic oligonucleotides, and/or fully protected syntheticoligonucleotides, the mixture having at least one partially or fullyprotected synthetic oligonucleotide with a protecting group covalentlybound thereto;

[0014] (b) providing an antibody microarray comprising a plurality ofantibodies immobilized on a substrate, each antibody specificallybinding to a synthetic oligonucleotide having an organic protectinggroup covalently bound thereto, which antibody does not bind to thesynthetic oligonucleotide when the organic protecting group is notcovalently bound thereto, the plurality of antibodies including at leastone antibody that is capable of binding at least one of the partially orfully protected synthetic oligonucleotides;

[0015] (c) contacting the synthetic oligonucleotide mixture with theantibodies immobilized on the antibody microarray such that at least oneof the protected synthetic oligonucleotides from the mixture is bound toat least one of the antibodies immobilized on the antibody microarray,wherein at least a portion of the oligonucleotides in the syntheticoligonucleotide mixture do not bind to the plurality of antibodies; and

[0016] (d) separating the antibody microarray and the unbound syntheticoligonucleotides, thus separating the unbound synthetic oligonucleotidesfrom any partially and/or fully protected synthetic oligonucleotide oroligonucleotides bound to the antibodies immobilized on the antibodymicroarray.

[0017] Yet another aspect of the present invention includes a method ofpreparing an oligonucleotide microarray using an antibody microarray.The method comprises the following steps:

[0018] (a) providing a synthetic oligonucleotide mixture prepared usingorganic protecting groups, the mixture comprising fully deprotectedsynthetic oligonucleotides, partially deprotected syntheticoligonucleotides, and/or fully protected synthetic oligonucleotides, themixture having at least one partially or fully protected syntheticoligonucleotide with a protecting group covalently bound thereto;

[0019] (b) providing an antibody microarray comprising a plurality ofantibodies immobilized on a first substrate, each antibody specificallybinding to a synthetic oligonucleotide having an organic protectinggroup covalently bound thereto, which antibody does not bind to thesynthetic oligonucleotide when the organic protecting group is notcovalently bound thereto, the plurality of antibodies including at leastone antibody that is capable of binding at least one of the partially orfully protected synthetic oligonucleotides;

[0020] (c) contacting the synthetic oligonucleotide mixture with theantibodies immobilized on the antibody microarray such that at least oneof the protected synthetic oligonucleotides from the mixture is bound toat least one of the antibodies immobilized on the antibody microarray,wherein at least a portion of the oligonucleotides in the syntheticoligonucleotide mixture do not bind to the plurality of antibodies;

[0021] (d) separating the antibody microarray and the unbound syntheticoligonucleotides, thus separating the unbound synthetic oligonucleotidesfrom any partially and/or fully protected synthetic oligonucleotide oroligonucleotides bound to the antibodies immobilized on the antibodymicroarray; and

[0022] (e) immobilizing the unbound synthetic oligonucleotides on asecond substrate to form an oligonucleotide microarray.

[0023] According to a further aspect of the present invention, anoligonucleotide microarray impregnated with antibodies is provided. Eachof the antibodies specifically binds to a synthetic oligomer having anorganic protecting group covalently bound thereto. The antibodies areimmobilized on a substrate along with an array of oligonucleotides. Insome embodiments, the antibodies may be encapsulated or one or moreblocking groups may be used to block the binding sites on the antibodiesuntil such time as detection of incomplete deprotection and/orinsufficient elongation is desired. In such embodiments, the antibodiesmay be contacted with the oligonucleotides on the oligonucleotidemicroarray when desired by releasing the antibodies from the capsules orremoving the blocking groups from the antibodies. A suitable assay maythen be performed to determine incomplete deprotection of the syntheticoligonucleotides and/or incomplete elongation of the syntheticoligonucleotides.

[0024] In summary, the antibodies, the antibody microarrays, theoligonucleotide microarrays impregnated with antibodies, and the methodsof the present invention are useful in immunoassays, such as for thequalitative and quantitative detection of protecting groups used inorganic synthetic processes, with particular application tooligonucleotides or peptides in research, therapeutics, diagnostics andbiomedical science. The present invention can be used in purificationtechniques, such as for the separation of final products from by-productcontaminants. The instant invention can be used in the course of qualitycontrol of oligonucleotide and peptide synthesis, such as in the qualitycontrol of drugs for gene therapy, antisense, antigene and control ofgene expression, in the quality control of biomedical polymers that maycontain protecting groups, and as probes or devices for purification andcharacterization of synthetic oligomers, particularly oligonucleotidesor peptides. The present invention advantageously provides highsensitivity in such quality control, purification, and immunoassaytechniques, even when using a low volume of antibodies. In addition, theuse of the present invention may be helpful in producing improvedoligonucleotide products such as, for example, improved DNA chips withlower amounts of incompletely deprotected/insufficiently elongated DNA.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 is a dot-blot immunoassay of monoclonal antibody 1H11,which selectively binds to oligoIbu-dG20mers.

[0026]FIG. 2 is a dot-blot immunoassay of monoclonal antibody 7H3, whichselectively binds to oligoBz-dC20mers.

[0027]FIG. 3 shows ELISA (FIG. 3A) and dot-blot (FIG. 3B) resultsdemonstrating specificity and detection sensitivity of a monoclonalantibody (mAb) of the commonly used protecting group, benzoyl (Bz), forthe chemical synthesis of nucleic acids. Partially deprotected oligomeroligo Bz-dC (center column) can be re-treated to remove the remainingprotecting groups, and re-tested with mAb (FIG. 3C). An RNA standardwith protecting groups Bz, ibu and ipr-Pac was synthesized and assayedfor identification of the protecting groups with the mAb against Bz(FIG. 3D).

[0028]FIG. 4 shows ELISA (FIG. 4A) and dot-blot (FIG. 4B) resultsdemonstrating specificity and sensitivity of a monoclonal antibody (mAb)and its detection of the commonly used protecting group, isobutryl(ibu), for the chemical synthesis of nucleic acids. Dot-blot assay withhigh amounts of DNA demonstrates that the ibu protecting group wasrecognized by the mAb no matter which nucleobase was protected (FIG.4C). Partially deprotected oligomer oligo Ibu-dg (center column) can bere-treated to remove the remaining protecting groups, and re-tested withmAb (FIG. 4D). An RNA standard with protecting groups Bz, ibu andipr-Pac was synthesized and assayed for identification of the protectinggroups with the mAb against ibu (FIG. 4E).

[0029]FIG. 5 shows ELISA (FIG. 5A) and dot-blot (FIG. 5B) resultsdemonstrating specificity and sensitivity of a monoclonal antibody (mAb)and its detection of the commonly used protecting group,isopropyl-phenoxyacetyl (ipr-Pac), for the chemical synthesis of nucleicacids. Partially deprotected oligomers oligo ibr-Pac-dG and oligo ibu-dG(columns second from left and forth from left, respectively) can bere-treated to remove the remaining protecting groups, and re-tested withmAb (FIG. 5C). An RNA standard with protecting groups Bz, ibu andipr-Pac was synthesized and assayed for identification of the protectinggroups with the mAb against ipr-Pac (FIG. 5D).

[0030]FIG. 6 shows a mAb dot-blot assay of protecting groupsdemonstrating the sensitivity and quantifiable response of thetechnology as related to HPLC. Dot-blot detection of Bz groups remainingon a standardized 20mer oligo dC molecule was analyzed (FIG. 6A) and aquantitation of the mAb response (FIG. 6B) was determined. The mAbresponse was analyzed with an increase in the amount of DNA on thedot-blot membrane (FIG. 6C). The column on the left is just theprotected Bz-dC 20mer. The column on the right is the protected Bz-dCtogether with a 2500-fold excess of the completely deprotected oligodC(Bz).

[0031]FIG. 7 shows a direct comparison of the mAb and HPLC detection ofBz in the pmole (FIG. 7A) and nmol range (FIG. 7B), respectively.

[0032]FIG. 8 shows a blind study demonstrating the detection ofremaining protecting groups in commercial samples. dA-dC oligos wereanalyzed with anti-Bz mAb (FIG. 8A) and dG-dT oligos were analyzed withanti-ipr-Pac mAb (FIG. 8B). The oligo dA-dC samples from companies #2and #6 were tested in higher amounts to confirm the presence of the Bzprotecting group (FIG. 8C). In addition, the samples were treated toremove the remaining protecting groups using a standard protocol. Theoligo dG-dT samples were assayed for the ipr-Pac protecting groups (FIG.8D). The samples were re-treated to remove remaining protecting groupsand re-analyzed as in (FIG. 8C).

[0033]FIG. 9 shows the production and analyses of polyclonal antibodyagainst the 5′terminal protecting group, dimethyltrityl (DMT).

[0034]FIG. 10 shows a substrate carrying different oligonucleotides ofthe same sequence, but with varying degrees of deprotection, that may beused as a testing standard to screen similar oligonucleotides of thesame sequence for varying degrees of protection or deprotection.

[0035]FIG. 11 illustrates an oligonucleotide array that may be screenedfor the presence of protecting groups or insufficient elongation withantibodies of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] 1. General Definitions.

[0037] “Antibody” as used herein refers to both monoclonal andpolyclonal antibodies, refers to antibodies of any immunoglobulin type(including but not limited to IgG and IgM antibodies), and includingantibody fragments that retain the hypervariable or binding regionsthereof. Antibodies may be of any species of origin, but are typicallymammalian (e.g., horse, rat, mouse, rabbit, goat). Antibodies may bebound to or immobilized on solid supports such as nitrocellulose,agarose, glass, organic polymers (“plastics”) and the like in accordancewith known techniques, and may be labeled with or joined to otherdetectable groups in accordance with known techniques.

[0038] “Binding” as used herein with respect to the selective binding ofan antibody to an oligomer has its usual meaning in the art. In general,to obtain useful discrimination in an immunoassay or an affinitypurification technique, the antibody should bind to the protectedoligomer at an affinity of at least about k_(d)=10⁻⁶, 10⁻⁷, or 10⁻⁸ M,and should bind to the unprotected oligomer at an affinity of notgreater than about k_(d)=10⁻², 10⁻³, or 10⁻⁴ M.

[0039] “Oligomer” as used herein refers to synthetic oligonucleotidesand synthetic oligopeptides, including synthetic oligomers in thenaturally occurring form such as DNA and RNA, and modified backbonechemistries as discussed below. Oligonucleotides are currently preferredin carrying out the present invention, and the instant invention isprimarily explained with reference to oligonucleotides herein. However,the methods and techniques described herein may also be applied tooligopeptides, oligosaccharides, etc. (i.e., any synthetically producedpolymer requiring protecting groups for synthesis).

[0040] “Nucleotide” as used herein refers to a subunit of anoligonucleotide comprising a pentose, a nitrogenous heterocyclic base(typically bound to the 1 position of the pentose), and a phosphate orphosphoric acid group (typically bound at the 5′ position of thepentose) but absent, or considered bound at the 3′ position, in the 5′terminal nucleotide of an oligonucleotide. These structures are wellknown. See, e.g., A. Lehninger, Biochemistry, 309-320). “Nucleoside”typically refers to a nucleotide, absent a phosphoric acid or phosphategroup.

[0041] “Protecting group” as used herein has its conventional meaning inthe art and refers to a chemical moiety, group or substituent that iscoupled, typically covalently coupled, to an atom in a molecule prior toa chemical reaction involving that molecule (typically in an organicsynthesis), so that the chemical reaction is averted at the atom towhich the protecting group is coupled. Typically, the protecting groupis then chemically removed from the intermediate molecule forpreparation of the final product, although removal techniques may not beentirely successful leading to only partial deprotection of the finalproduct (i.e., the presence of at least one protecting group remainingon that molecule). Protecting groups may be intentionally left on amolecule for purposes of generating or testing an antibody as describedherein.

[0042] “Deprotection” or “deprotected” as used herein refers to theabsence of protecting groups employed during chemical oligonucleotidesynthesis from a molecule. Such protecting groups are described below.The presence of such a protecting group may indicate insufficientelongation of the oligonucleotide, when the protecting group is chainterminating. Chemically synthesized oligonucleotides are ideally fullydeprotected, but the present invention is employed to detect partial orincomplete deprotection of such oligonucleotides (that is, the presenceof at least one protecting group as described below in theoligonucleotide).

[0043] “Base” as used herein with respect to oligonucleotides refers toa nitrogenous heterocyclic base which is a derivative of either purine(e.g., adenine, guanine) or pyrimidine (e.g., uracil, thymine,cytosine). Pyrimidine bases are bound to the pentose by the 1 ringnitrogen; Purine bases are bonded to the pentose by the 9 ring nitrogen.Preferred bases are those that contain a free amino group, such asguanine, adenine, and cytosine (the protecting group is then covalentlybound to the free amino group by substitution of one, or both, of thehydrogens on the free amino group). However, the present invention maybe used with any purine or pyrimidine base, whether standard ormodified/rare, that contains a free amino group for protection, or othergroup requiring protection during synthesis thereof in anoligonucleotide. Examples of standard and modified/rare bases are thosefound in the nucleosides set forth in Table 1 below. TABLE 1 Standardand modified nucleosides and their standard abbreviations. abbreviationbase U uridine C cytidine A adenosine G guanosine T thymidine ?A unknownmodified adenosine m1A 1-methyladenosine m2A 2-methyladenosine i6AN⁶-isopentenyladenosine ms2i6A 2-methylthio-N⁶-isopentenyladenosine m6AN⁶-methyladenosine t6A N⁶-threonylcarbamoyladenosine m6t6AN⁶-methyl-N⁶-threonylcarbomoyladenosine ms2t6A2-methylthio-N⁶-threonylcarbamoyladenosine Am 2′-O-methyladenosine IInosine m1I 1-methylinosine Ar(p) 2′-O-(5-phospho)ribosyladenosine io6AN⁶-(cis-hydroxyisopentenyl)adenosine ?C Unknown modified cytidine s2C2-thiocytidine Cm 2′-O-methylcytidine ac4C N⁴-acetylcytidine m5C5-methylcytidine m3C 3-methylcytidine k2C lysidine f5C 5-formylcytidinef5Cm 2′-O-methyl-5-formylcytidine ?G unknown modified guanosine Gr(p)2′-O-(5-phospho)ribosylguanosine m1G 1-methylguanosine m2GN²-methylguanosine Gm 2′-O-methylguanosine m22G N²N²-dimethylguanosinem22Gm N²,N²,2′-O-trimethylguanosine m7G 7-methylguanosine fa7d7Garchaeosine Q queuosine manQ mannosyl-queuosine galQgalactosyl-queuosine Yw wybutosine o2yW peroxywybutosine ?U unknownmodified uridine mnm5U 5-methylaminomethyluridine s2U 2-thiouridine Um2′-O-methyluridine s4U 4-thiouridine ncm5U 5-carbamoylmethyluridinemcm5U 5-methoxycarbonylmethyluridine mnm5s2U5-methylaminomethyl-2-thiouridine mcm5s2U5-methoxycarbonylmethyl-2-thiouridine cmo5U uridine 5-oxyacetic acidmo5U 5-methoxyuridine cmnm5U 5-carboxymethylaminomethyluridine cmnm5s2U5-carboxymethylaminomethyl-2-thiouridine acp3U3-(3-amino-3-carboxypropyl)uridine mchm5U5-(carboxyhydroxymethyl)uridinemethyl ester cmnm5Um5-carboxymethylaminomethyl-2′-O-methyluridine ncm5Um5-carbamoylmethyl-2′-O-methyluridine D Dihydrouridine ψ pseudouridinem1ψ 1-methylpseudouridine ψm 2′-O-methylpseudouridine m5U ribosylthyminem5s2U 5-methyl-2-thiouridine m5Um 5,2′-O-dimethyluridine

[0044] See Sprinzl et al., Nucleic Acids Res. 26, 148 (1998).

[0045] Applicants specifically intend that the disclosures of all UnitedStates patent references cited herein be incorporated by referenceherein in their entirety.

[0046] 2. Protecting Groups.

[0047] The particular protecting group will depend upon the oligomerbeing synthesized and the methodology by which that oligomer issynthesized.

[0048] For the synthesis of oligonucleotides, suitable protecting groupsinclude alkyl, aryl, alkylaryl, arylalkyl groups, which may contain oneor more hetero atoms such as N, O, or S, and which may be substituted orunsubstituted (e.g., a carbonyl group). Examples of protecting groupsinclude, but are not limited to, the following: acetyl; isobutyryl;2-(t-butyldiphenyl-silyloxymethyl)benzoyl; naphthaloyl;iso-butyryloxycarbonyl; levulinyl; fluorenylmethoxycarbonyl;2-nitrothiophenyl; 2,2,2-trichloro-t-butoxycarbonyl, ethoxycarbonyl;benzyloxycarbonyl; p-nitrophenyl-ethyloxycarbonyl;N′N-dimethylformamidine; formyl; benzoyl, toluyl;2,4-6-trimethylbenzoyl; anisoyl; 2,4-dimethylphenyl;2,4,6-trimethylphenyl; triphenylthiomethyl; pivoloiloxymethyl;t-butoxycarbonyl; p-nitrophenylethyl; methoxyethoxymethyl;butylthiocarbonyl; 2-methyl-pyridine-5-yl; 2-nitrothiophenyl;2,4-dinitrothiophenyl; 2-nitro-4-methylthiophenyl;p-nitrophenylsulphonylethyl; 5-chloro-8-hydroxyquinoline; thiophenyl;β-cyanoethyl; phenylethyl; p-nitrophenylethyl; pyridylethyl;2-N-methylimidazolylphenyl; methyl; allyl; trichloroethyl; dibenzoyl;p-nitrophenylethoxycarbonyl; benzoyl and substituted derivativesthereof; 2(acetoxymethyl) benzoyl; 4,4′,4″-tris-(benzyloxy)trityl;5-methylpyridyno-2-yl; phenylthioethyl; dipehylcarbamoyl;3,4-dimethoxybenzyl; 3-chlorophenyl; 2-nitrophenyl;9-pnenylxanthen-9-yl; 9-(p-methoxyphenyl)xanthen-9-yl;9-(p-ocatadecyloxyphenyl)xanthen-9-yl; “bridged” bis-dimethoxytritylgroups; phthaloyl; succinyl; benzensulphonylethoxycarbonyl;4,4′,4″-tris(bevulinyloxy)trityl; p-phenylazophenyloxycarbonyl;o-substituted benzoyl; 4,4′4″-tris-(4,5-dichlorophalimidin)trityl;levelinyl; alkyloxy and aryloxyacetyl; 1,3-benzodithiol-2-yl;tetrahydrofuranyl; [2-(methylthio)phenyl]thiomethyl;1-(2-chloroethyoxy)ethyl; 1-[(2-fluorophenyl]4-methoxy piperidin-4-yl;4-methoxytetrahydropyran-4-yl; (1-methyl-1-methoxy)ethyl;tetrahydropyranyl; 3-methoxy-1,5-dicarbomethoxypentam-3-yl;2-nitrobenzyl; benzyl; 4-nitrophenylethyl-sulphonyl;t-butyldimethylsilyl; 4-methoxybenzyl; 3,4-dimethoxybenzyl;9-p-methoxyphenylthioxanthen-9-yl; compounds of the formula X¹X²X³C—,wherein X¹, X², and X³ are each independently selected from the groupconsisting of phenyl, p-monomethoxyphenyl, o-monomethoxphenyl, biphenyl,p-fluoropnehyl, p-chlorophenyl, p-methylphenyl, p-nitrophenyl, etc.

[0049] 3. Oligonucleotides.

[0050] Synthetic oligonucleotides that contain protecting groups and maybe used to carry out the present invention include both the naturallyoccurring forms such as DNA and RNA, and those with modified backbonechemistries, such as poly (phosphate derivatives) such as phosphonates,phosphoramides, phosphonamides, phosphites, phosphinamides, etc., poly(sulfur derivatives) e.g., sulfones, sulfonates, sulfites, sulfonamides,sulfenamides, etc. It is noted that antibodies of the invention may becharacterized by their selective binding to particular “reagent” or“benchmark” oligonucleotides, but the same antibodies may also bind to avariety of other oligonucleotides (e.g., longer or shorter nucleotides)or other compounds that contain the same protecting group.

[0051] For example, an oligonucleotide to which the antibody selectivelybinds may consist of from 3 to 20 nucleotides, and wherein one of saidnucleotides is a protected nucleotide according to Formula (I) below:

[0052] wherein:

[0053] R is H or a protecting group, such as dimethoxytrityl; subject tothe proviso that R is a covalent bond to an adjacent nucleotide whensaid protected nucleotide is not a 5′ terminal nucleotide in saidoligonucleotide;

[0054] R¹ is H or a protecting group such as β-cyanoethyl; subject tothe proviso that R¹ is a covalent bond to an adjacent nucleotide whensaid protected nucleotide is not a 3′ terminal nucleotide in saidoligonucleotide;

[0055] R² is H or —OR³;

[0056] R³ is H or a protecting group such as tert-butyldimethylsilyl;

[0057] Base is a purine or pyrimidine base; and

[0058] R⁴ is a protecting group substituted for a hydrogen on an aminogroup of the base, such as a protecting group selected from the groupconsisting of acetyl (Ac), benzoyl (Bz), dimethylformamidine (dmf),isobutyrl (Ibu), phenoxyacetyl (Pac), and isopropyl-phenoxyacetyl(Ipr-pac), subject to the proviso that R⁴ is not present in Formula (I)when R, R¹, or R³ is a protecting group or when the base does notinclude an amino group; and

[0059] further subject to the proviso that only one of R, R¹, R³, and R⁴is a protecting group and the others of R, R¹, R³, and R⁴ are notprotecting groups.

[0060] In one particular embodiment of the foregoing, the antibody maybe one that selectively binds to an oligonucleotide that consists offrom 3 to 20 nucleotides and has a 5′ nucleotide, and wherein said 5′nucleotide is a protected nucleotide according to Formula (IA):

[0061] wherein:

[0062] R⁵ is a protecting group such as dimethoxytrityl;

[0063] R⁶ is a covalent bond to an adjacent nucleotide;

[0064] R⁷ is H or —OH; and

[0065] Base is a purine or pyrimidine base.

[0066] In another particular embodiment of the foregoing, the antibodymay be one that selectively binds to an oligonucleotide that consists offrom 3 to 20 nucleotides and has a 3′ nucleotide, and wherein said 3′nucleotide is a protected nucleotide according to Formula (IB):

[0067] wherein:

[0068] R⁸ is a covalent bond to an adjacent nucleotide;

[0069] R⁹ is a protecting group such as β-cyanoethyl;

[0070] R¹⁰ is H or —OH; and

[0071] Base is a purine or pyrimidine base.

[0072] In another particular embodiment of the foregoing, the antibodymay be one that selectively binds to an oligonucleotide that consists offrom 3 to 20 nucleotides, and wherein one of said nucleotides is aprotected nucleotide according to Formula (IC):

[0073] wherein:

[0074] R¹¹ is a covalent bond to an adjacent nucleotide;

[0075] R¹² is a covalent bond to an adjacent nucleotide;

[0076] R¹³ is —OR¹⁴;

[0077] R¹⁴ a protecting group such as tert-butyldimethylsilyl; and

[0078] Base is a purine or pyrimidine base.

[0079] In still another particular embodiment of the foregoing, theantibody may be one that selectively binds to an oligonucleotide thatconsists of from 3 to 20 nucleotides, and wherein one of saidnucleotides is a protected nucleotide according to Formula (ID):

[0080] wherein:

[0081] R¹⁵ is a covalent bond to an adjacent nucleotide;

[0082] R¹⁶ is a covalent bond to an adjacent nucleotide;

[0083] R¹⁷ is H or —OH;

[0084] Base is a purine or pyrimidine base; and

[0085] R¹⁸ is a protecting group bonded to an amino group of said base,such as acetyl, benzoyl, dimethylformamidine, isobutyryl, phenoxyacetyl,and isopropyl-phenoxyacetyl.

[0086] Thus, examples of protected bases that may be employed in thestructures shown above include, but are not limited to, adenine,guanine, and cytosine, as follows:

[0087] wherein Y¹ and Y² are both H in an unprotected base, and eitheror both Y¹ and/or Y² are a protecting group as described above (e.g.Pac, Ipr-pac, Ibu, Bz, Ac, dmf) for a protected base. Likewise, modifiednucleosides have protecting groups at the modifications that arechemically reactive.

[0088] In one embodiment of the invention, the oligonucleotides arepeptide nucleic acids, and the protecting groups are those protectinggroups employed in the synthesis of peptide nucleic acids, including butnot limited to those described in U.S. Pat. No. 6,133,444.

[0089] In still another particular embodiment of the foregoing, theantibody may be one that selectively binds to an oligonucleotide thatconsists of from 3 to 20 nucleotides, and wherein one of saidnucleotides is protected with a photolabile protecting group, includingbut not limited to those described in U.S. Pat. Nos. 5,744,101 and5,489,678.

[0090] 4. Antibodies.

[0091] As noted above, the present invention provides antibodies (e.g.,a monoclonal or polyclonal antibody) that specifically bind to asynthetic oligonucleotide having an organic protecting group covalentlybound thereto, which antibody does not bind to said syntheticoligonucleotide when said organic protecting group is not covalentlybound thereto.

[0092] The antibody may be provided immobilized on (or bound to) a solidsupport in accordance with known techniques, or may be provided in afree, unbound form (e.g., lyophilized, frozen, in an aqueous carrier,etc.). Whether or not an antibody is immobilized will depend upon theparticular immunoassay or affinity purification technique in which theantibody is used, and is determined by the known parameters for suchtechniques. Similarly, the antibody may be bound to or conjugated withsuitable detectable groups, such as an enzyme (e.g., horseradishperoxidase), a member of a binding pair such as biotin or avidin, aradioactive group or a fluorescent group such as green fluorescentprotein, also in accordance with known techniques, typically dependingupon the immunoassay format in which the antibody is used.

[0093] 5. Immunoassay Methods.

[0094] The present invention provides a method for detecting incompletedeprotection of a synthetic oligonucleotide (including aborted sequencesthat still contain a protecting group) by immunoassay. In general, suchan immunoassay comprises the steps of: (a) contacting a syntheticoligonucleotide to an antibody as described above, and then (b)detecting the presence or absence of binding of said antibody to saidoligonucleotide, the presence of binding indicating incompletedeprotection of said synthetic oligonucleotide. Any suitable assayformat can be employed, including heterogeneous and homogeneousimmunoassays. For example, the immunoassay may be an immunodot-blotassay, or may be a sandwich assay. The oligonucleotides being tested fordeprotection may be in any suitable form, such as in solution orimmobilized on a solid support.

[0095] In a preferred embodiment, the detection method employs a “dipstick” or the like, in which binding of the antibody to the testoligonucleotide is compared to binding of the antibody to a set of knownoligonucleotides, all immobilized on a common solid support. Such anarticle, as illustrated in FIG. 10, useful for determining incompletedeprotection of a synthetic oligonucleotide in an immunoassay,comprises: (a) a solid support (e.g., a nitrocellulose strip) 25 havinga surface portion, said surface portion having at least two separatediscrete regions 26, 27 formed thereon; (b) a first oligonucleotidebound to one of said separate discrete regions, said firstoligonucleotide having a protecting group bound thereto (e.g., at leastone protecting group); and (c) a second oligonucleotide bound to anotherof said separate discrete regions, said second oligonucleotide nothaving said protecting group bound thereto; wherein the nucleotidesequence of said first and second oligonucleotides are the same. In apreferred embodiment, the article further comprises (d) a thirdoligonucleotide bound to another of said separate discrete regions 28;said third oligonucleotide also having said protecting group bound tosaid first oligonucleotide bound thereto; wherein said thirdoligonucleotide is partially deprotected (i.e., has a number ofprotecting groups covalently bound thereto which is intermediate betweenthat bound to the first and second oligonucleotide, e.g., at least one,two three or four more protecting groups than the first oligonucleotide,up to at least 10, 20 or more protecting groups than the firstoligonucleotide); and wherein the nucleotide sequence of said first,second, and third oligonucleotides are the same. Of course, still moreoligonucleotides carrying varying numbers of protecting groups may beincluded on the substrate in additional separate and discrete locations,if desired. The discrete regions to which the separate oligonucleotidesare bound may be in any form, such as dots.

[0096] 6. Affinity Purification Methods.

[0097] In addition to immunoassays, the present invention also providesaffinity purification techniques for the separation of fully deprotectedoligonucleotides from partially deprotected (including fully protected)oligonucleotides (e.g., both oligonucleotides that have been subjectedto a deprotection process to remove the protecting group, andoligonucleotides that have not). Such a procedure typically comprises(a) contacting a mixture of protected and fully deprotected syntheticoligonucleotides to antibodies as described above, wherein the protectedsynthetic oligonucleotides have the organic protecting group for whichthe antibody is selective covalently bound thereto, so that theprotected synthetic oligonucleotides bind to the antibody; and then (b)separating said antibodies from said fully deprotected oligonucleotides.The antibody may be immobilized on a solid support to facilitateseparation. The protected synthetic oligonucleotide may be a partiallyprotected synthetic oligonucleotide, or a fully protected syntheticoligonucleotide that has not undergone deprotection. Any separationformat may be used, including but not limited to affinitychromatography.

[0098] 7. Production of Antibodies.

[0099] A method of making an antibody that specifically binds to asynthetic oligonucleotide having an organic protecting group covalentlybound thereto, which antibody does not bind to the said syntheticoligonucleotide when said organic protecting group is not covalentlybound thereto, comprises the steps of: (a) synthesizing the syntheticoligonucleotide on a solid particulate support (and preferablycovalently bound thereto, e.g., with a succinyl linker) with the organicprotecting group covalently bound to said synthetic oligonucleotide; andthen, without removing the oligonucleotide from said solid support; and(b) immunizing an animal with the synthetic oligonucleotide bound to thesolid support in an amount sufficient to produce the antibody. Inaddition, a single nucleotide can be bound to the solid particulatesupport with the organic protecting group bound thereto, and used asdescribed hereinabove.

[0100] The synthesis step may be carried out on the solid support inaccordance with known techniques. The solid support may be inparticulate form prior to synthesis, or may be fragmented into particlesafter synthesis. In general, the solid supports are beads, which may becompletely solid throughout, porous, deformable or hard. The beads willgenerally be at least 10, 20 or 50 to 250, 500, or 2000 μm in diameter,and are most typically 50 to 250 μm in diameter. Any convenientcomposition can be used for the solid support, including cellulose,pore-glass, silica gel, polystyrene beads such as polystyrene beadscross-linked with divinylbenzene, grafted copolymer beads such aspolyethyleneglycol/polystyrene, polyacrylamide beads, latex beads,dimethylacrylamide beads, composites such as glass particles coated witha hydrophobic polymer such as cross-linked polystyrene or a fluorinatedethylene polymer to which is grafted linear polystyrene, and the like.Where separate discrete solid supports such as particles or beads areemployed, they generally comprise from about 1 to 99 percent by weightof the total reaction mixture.

[0101] In a preferred embodiment, the synthesizing step is followed bythe step of fragmenting the solid support (e.g., by crushing) prior tothe immunizing step. Polyclonal antibodies may be collected from theserum of the animal in accordance with known techniques, or spleen cellsmay be collected from the animal, a plurality of hybridoma cell linesproduced from the spleen cells; and then a particular hybridoma cellline that produces the antibody isolated from the plurality of hybridomacell lines.

[0102] A particular protocol for the production of antiserum/polyclonalantibodies and monoclonal antibodies against protecting groups used innucleic acid and other synthesis typically involves the following steps:(a) preparation of oligonucleotides and others that contain or do notcontain protecting groups; (b) immunization of animals with thosepreparations; (c) screening of animals to identify those that exhibitantibodies against protecting groups; (d) production of monoclonalantibody by classical fusion method; (e) optionally, production ofscFab, Fab fragments and whole antibody molecules by antibodyengineering; and (f) evaluation and characterization of monoclonalantibodies against the protecting groups. Each of these steps isdiscussed in greater detail below.

[0103] Synthetic oligonucleotides that contain protecting groups can besynthesized in a variety of ways known to those skilled in the art. Forexample, protecting groups can be attached to individual nucleotidesthat are linked to controlled pore glass (CPG) beads. An example is:

[0104] CPG bead---dT (only with DMT group).

[0105] In the alternative, protecting groups may be attached tooligonucleotide chains that are linked to CPG beads. Examples include:

[0106] Pac-dA---Pac-dA---CPG beads with Bz-dC and Ibu-dG;

[0107] Ipr-Pac-dG---Ipr-Pac-dG---CPG beads with Bz-dC and Ibu-dG;

[0108] Ac-dC---Ac-dC---CPG beads with Bz-dC and Ibu-dG;

[0109] dmf-G---dmf-G---CPG beads with Bz-dC and Ibu-dG; and

[0110] mixtures of the four oligonucleotides described above.

[0111] In another alternative, protecting groups may be attached tooligonucleotide chains that are partially deprotected (the procedure fordeprotection will be described below). Examples include:

[0112] Poly dT20mers (only with DMT group);

[0113] Poly dT20mers (only with cyanoethyl groups);

[0114] Poly Ibu-dG 20mers (partially deprotected);

[0115] Poly Ipr-Pac-dG 20mers (partially deprotected);

[0116] Poly Bz-dC 20mers (partially deprotected);

[0117] Poly Pac-dA 20mers (partially deprotected); and

[0118] Poly Ac-dC 20mers (partially deprotected).

[0119] Synthetic oligonucleotides prepared as described herein may bepartially deprotected as follows: (a) add 30% ammonium hydroxidesolution to synthetic polynucleotides, then incubate at room temperaturefor different time periods (5, 10 and 30 min); (b) take the ammoniumsolution of treated oligomers and add into 1:1 diluted acetic acidpre-cooled at 4° C. and according to 1:4 ratio of ammonium to aceticacid; (c) keep samples in ice bath for 30 min; (d) dry samples withspeed-Vac; (e) dissolve the dried pellets in water; (f) desalt sampleswith Sephadex G-25 column; (g) dry samples with speed-Vac; and (h)dissolve the desalted samples in water.

[0120] Synthetic oligonucleotides prepared as described herein may becompletely deprotected by any suitable technique. One particulartechnique is as follows: (a) add 30% ammonium hydroxide solution tosynthetic oligonucleotides, then incubate at 65° C. for 6 hrs; (b) drysamples with speed-Vac; (c) dissolve the dried pellets in water; (d)desalt samples with Sephadex G-25 column; and (e) dry samples withspeed-Vac; (f) redissolve desalted samples in water.

[0121] Partially and completely deprotected oligonucleotides may becharacterized for further use or to verify procedures by any suitablemeans, including but not limited to gel electrophoresis, urea-acrylamidegel electrophoresis, 5′end labeling with T4 polynucleoide kinase, HPLCanalysis, mass spectrometry, etc.

[0122] Suitable animals can be immunized with the oligonucleotidesdescribed above by parenteral injection of the oligonucleotide in asuitable carrier, such as sterile saline solution. Injection may be byany suitable route, including but not limited to subcutaneous,intraperitoneal, intravenous, intraarterial, intramuscular, etc.Suitable animals are typically mammals, including mice, rabbits, rats,etc.

[0123] In a particular embodiment, for the production of monoclonalantibodies, young female BALB/c mice are used, and the time course ofinjection of the antigen material is: first day initial injection 14thday first boosting 28th day second boosting  4 day before fusion finalboosting

[0124] Additional injections may be employed if desired. The antigenamount may be 50 μg or 100 μg of oligonucleotides unprotected (forcontrol antibody) or protected, for each mouse per time. When, aspreferred, beads or other solid support used as the support foroligonucleotide synthesis are injected into the animal, the beads orparticles are suspended in water, then injected into mice. If anucleotide solution is used, then the solution is mixed with complete orincomplete Freund's adjuvant and injected into mice.

[0125] Polyclonal antibodies can be harvested from animals immunized orinnoculated as described above in accordance with known techniques, orspleen cells harvested from the animals, hybridoma cell lines producedfrom the spleen cells, and the hybridoma cell lines screened for theproduction of desired antibodies, also in accordance with knowntechniques.

[0126] Oligonucleotides that contain or do not contain biotin moleculesat 3′ or 5′ ends (for ELISA assay as described below) may be synthesizedin accordance with standard techniques. Examples are:

[0127] Poly Ibu-dG 20 mers (with or without biotin);

[0128] Poly Ibu-dA 20 mers (with or without biotin);

[0129] Poly Ibu-dC 20 mers (with or without biotin);

[0130] Poly Ipr-Pac-dG 20 mers (with or without biotin);

[0131] Poly Bz-dC 20 mers (with or without biotin);

[0132] Poly Bz-dA 20 mers (with or without biotin);

[0133] Poly dT 20 mers (with or without biotin);

[0134] Poly Pac-dA 20 mers (with or without biotin);

[0135] Poly Ac-dC 20 mers (with or without biotin); and

[0136] Poly dmf-G 20 mers (with or without biotin).

[0137] Antibodies produced as described above may be characterized byany suitable technique to determine the binding properties thereof,including but not limited to Western blot and immunodot-blot.

[0138] In addition to the use of polyclonal and monoclonal antibodies,the present invention contemplates the production of antibodies byrecombinant DNA, or “antibody engineering” techniques. For example, mRNAisolated from hybridoma cells may be used to construct a cDNA libraryand the sequence encoding whole antibody or antibody fragments (e.g.,scFab or Fab fragments) isolated and inserted into suitable expressionvector, and the expression vector inserted into a host cell in which theisolated cDNA encoding the antibody is expressed.

[0139] Monoclonal Fab fragments may be produced in Escherichia coli byrecombinant techniques known to those skilled in the art. See, e.g., W.Huse, Science 246, 1275-81 (1989).

[0140] 8. Screening of Antibodies.

[0141] Screening sera and hybridoma cell culture media for protectinggroup specific antibodies may be carried out as follows:

[0142] A. Sera

[0143] 1. Pre-immune (prior to immunization) sera are collected bystandard means from the mice to be inoculated with protecting groupconjugated to a solid support (directly or through an oligomer).

[0144] 2. Post-innoculation sera are also collected.

[0145] 3. An ELISA assay is performed in which the specific protectinggroup remains on a biotinylated oligonucleotide conjugated to themicrotiter plate. Other microtiter plate wells contain control oligomersthat have no protecting groups, or oligonucleotides with otherprotecting groups. The secondary antibody is a goat anti-mouse IgG witha conjugated phosphotase for visualization of antibody.

[0146] 4. Those mice that have positive activity against the specificprotecting group are boosted and sacrificed for the production ofhybridomas.

[0147] B. Hybridoma Cell Culture Media

[0148] 1. Approximately 1000 cultures are generated from each spleenhybrid cell production.

[0149] 2. Cultures are grown in microtiter plate wells, 96 well plates.

[0150] 3. Culture medium is removed from each well and used in ELISAassays as described above in which each of the ˜1000 microtiter platewells contain the protected oligonucleotide conjugated to the plate.

[0151] 4. Those cultures producing antibody that has positive activityare transferred to larger culture wells, 24 well microtiter plates.

[0152] 5. Culture media from the larger cultures are re-tested foractivity against the protecting group and are also assayed forspecificity; i.e. controls of no protecting group and of otherprotecting groups.

[0153] 6. Those cultures that are positive are cloned out (diluted),re-tested and cloned out again to the point that each final culture mustbe the result of one cell; i.e., mono-culture. Media from these finalcultures are thoroughly assessed for specificity and affinity.Specificity and affinity are assessed using a dot-blot assay.

[0154] C. Dot-Blot Assays in Lieu of ELISA Assays

[0155] 1. Antibodies against some protecting groups are not tractable tobeing tested in the microtiter plate well environment and must be testedusing a dot-blot assay. One example is the 5′-terminal protecting group,dimethyl-trityl (DMT).

[0156] 2. The Dot-blot assay on a nitrocellulose membrane isaccomplished as described elsewhere in the application for mostpurposes. However, this is not possible in assessing antibody productionby ˜1000 microtiter well cultures with little media available. Thus, anovel adaptation has been developed.

[0157] a) The protected oligonucleotide is attached in dots to thenitrocellulose using UV-crosslinking. With DMT, the presence of the5′-DMT on the membrane is confirmed by treatment of a dot with mildacid—the dot turns yellow-orange. The presence of the 3′-biotin can beconfirmed with a commercial avidin stain.

[0158] b) The membrane is blocked (see dot-blot assay).

[0159] c) The dry membrane dots are carefully marked (pencil) and“punched” out of the membrane.

[0160] d) Individual dots are added to the cell culture media inindividual microtiter plate wells and incubated.

[0161] e) The individual dots are removed and passed on through thewashing, secondary antibody, phosphotase reaction and color developmentusing microtiter plate wells with the appropriate reagents.

[0162] f) Those dots that are positive are related back to the originalmicrotiter plate well cultures from which the small amount of culturemedia was obtained.

[0163] g) Further culturing and cloning is accomplished as described inB.

[0164] 9. Testing of Microarrays.

[0165] The present invention may be used to test or screenoligonucleotides that are immobilized on a solid support such as amicroarray for insufficient deprotection or elongation of theoligonucleotides synthesized thereon.

[0166] Solid supports used to carry out the present invention aretypically discrete solid supports. Discrete solid supports may bephysically separate from one another, or may be discrete regions on asurface portion of a unitary substrate. Such “chip-type” or “pin-type”solid supports are known. See, e.g., U.S. Pat. No. 5,143,854 to Pirrung;U.S. Pat. No. 5,288,514 to Ellman (pin-based support); U.S. Pat. No.5,510,270 to Fodor et al. (chip-based support). Additional non-limitingexamples of oligonucleotide arrays which may be used to carry out thepresent invention, and methods of making the same, include but are notlimited to those described in U.S. Pat. Nos. 5,631,734; 5,599,695;5,593,839; 5,578,832; 5,510,270; 5,571,639; 6,056,926; 5,445,934; and5,703,223. Such devices maybe used as described therein to carry out theinstant invention.

[0167] The solid support or substrate from which the array is formed maybe comprised of any suitable material, including silicon. Theoligonucleotides may be polymerized or grown in situ from monomers (orindividual nucleotides) in situ on the microarray (in which case none ofthe currently available techniques for detecting protecting groups wouldbe useful for detecting incomplete deprotection or elongation of theoligonucleotides on the array, as one cannot pass the solid supportthrough an analytical device) or the oligonucleotides may be polymerizedseparately and then linked to the appropriate regions of the solidsupport. The array may include any number of different oligonucleotidesin different separate and discrete regions thereon, examples includingarrays of at least 1,000, at least 2,000, at least 10,000, or at least20,000 different oligonucleotides in different separate and discreteregions.

[0168] In general, a method of screening an oligonucleotide array forinsufficient deprotection or insufficient elongation of oligonucleotidestherein comprises the steps of:

[0169] (a) providing an oligonucleotide array as described above;

[0170] (b) providing an antibody as described above (that is, anantibody that specifically binds to a synthetic oligonucleotide havingan organic protecting group covalently bound thereto, which antibodydoes not bind to said synthetic oligonucleotide when said organicprotecting group is not covalently bound thereto). Preferably theantibody is one that specifically binds to an oligonucleotide having aprotecting group, where the protecting group was employed in the courseof the organic synthesis of oligonucleotides carried by that array.Then;

[0171] (c) contacting said oligonucleotide array to said antibody tothereby detect the presence of insufficient deprotection or insufficientelongation of oligonucleotides therein. Such detection, which may bequalitative or quantitative, may be carried out by any suitableimmunoassay technique as described above.

[0172] In the method, steps (b) to (c) may be repeated at least once,with a different antibody on each repetition, so that a plurality ofdifferent protecting groups which may be present on oligonucleotides inthe array may be detected.

[0173] Preferably, once insufficient deprotection (the presence ofprotecting groups) in oligonucleotides in one or more (e.g., plurality)of the separate and discrete regions is detected, the method furthercomprises generating a record or indicia recording the presence ofinsufficient deprotection or insufficient elongation of oligonucleotidesin the least one separate and discrete location (or plurality ofseparate and discrete locations) on the array. The indicia may be aqualitative or quantitative indicia of insufficient deprotection(including insufficient elongation).

[0174] The foregoing methods provide a correctable oligonucleotide arrayas illustrated in FIG. 11. The array comprises, in combination:

[0175] (a) a substrate 30 having a plurality of differentoligonucleotides immobilized thereon, with the differentoligonucleotides immobilized in different separate and discretelocations 31 on said substrate; and

[0176] (b) a plurality of indicia associated with said array, theseindicia recording the presence of insufficient deprotection orinsufficient elongation of a plurality of different oligonucleotides,said different oligonucleotides located in separate and discretelocations on said array. These indicia may be printed in a region of thearray 32 by a technique such a microlithography, printed on conventionalmedium such as paper and shipped with the array, stored in a memory ormemory device connected to or formed on the array chip (which may beincorporated at location 32), provided in a separate data or computerfile which may be provided on a computer-readable medium such as afloppy diskette or CD-ROM, stored on a web site on the world wide webfor downloading by the end user of the array, etc. When the indicia areprovided in a separate data file, the array preferably further includesan identifier such as a code number formed on, connected to orassociated with the array (e.g., printed on a package containing thearray, or on an information sheet packaged with the array, and/orprinted directly on the array). The identifier may then be associatedwith the separate indicia (e.g., printed on a data sheet, used as apass-word, file identifier and/or access code for the computer file,etc.) to insure the correct indicia containing the record ofinsufficient deprotection and/or elongation are ultimately associatedwith the array by the ultimate end user of the array.

[0177] A data device or memory device connected to the array may becarried out in accordance with known techniques, as described in U.S.Pat. Nos. 5,925,562; 6,017,496; 5,751,629; and 5,741,462, and suchdevices used as described therein to carry out the instant invention.

[0178] The end user of the array may utilize the indicia described aboveto compensate for insufficient deprotection or insufficient elongationof oligonucleotides on said array in a method comprising:

[0179] (a) providing a substrate as described above.

[0180] (b) providing at least one, or a plurality of, indicia associatedwith said array as described above.

[0181] (c) providing a test compound. The test compound may be a memberof a library of test compounds, and may be any suitable compound such asa protein, peptide or oligonucleotide (e.g., a DNA or RNA, such asmRNA); and then

[0182] (d) detecting the binding of said test compound to at least oneof said plurality of different oligonucleotides (e.g., by contacting thetest compound to the array); and then

[0183] (e) detecting determining the degree of binding (including simplythe presence or absence of binding) of the test compound to one or moreoligonucleotides on the array from (i) said detected binding and (ii)said indicia recording the presence of insufficient deprotection orinsufficient elongation. Thus, insufficient deprotection or insufficientelongation of oligonucleotides in one or more locations in the array maybe compensated for during the determining step. Such compensation may beachieved by any means, including ignoring particular separate anddiscrete regions on the array (e.g., in favor of other separate anddiscrete regions of the array that contain the same oligonucleotide). Inanother example, if one or more locations contain insufficientdeprotection or elongation such that binding to those locations isreduced, the binding data derived from an experiment with that array canbe adjusted upwards for those locations to indicate greater binding thanthat which would otherwise be indicated without the control madepossible by the recorded indicia. The detecting or determining step maybe carried out by any suitable means, such as generating a colorindication of degree of binding, generating a numeric indication ofdegree of binding, generating a graphic or other symbolic indication ofdegree of binding, etc. The degree of binding may be an indication ofbinding is binding affinity, binding amount, or both binding affinityand binding amount, but is typically an indication of the amount of testcompound that binds to a particular separate and discrete region of thearray.

[0184] 10. Antibody Microarrays.

[0185] The present invention includes antibody microarrays or “antibodychips” formed with a plurality or an array of antibodies immobilized ona substrate. Each of the antibodies used on the antibody microarrayspecifically binds to a synthetic oligomer (such as a syntheticoligonucleotide) having an organic protecting group bound thereto, asdescribed above. Such an antibody chip may be used to detect incompletedeprotection of synthetic oligomers and/or separate protected syntheticoligomers from deprotected synthetic oligomers. Such an antibody chipmay also be used to detect incomplete elongation of synthetic oligomersand/or separate full-length synthetic oligomers from incompletesynthetic oligomers (e.g., when the protecting group is chainterminating). The antibody chips according to the present invention andthe methods of using them described below may be useful in highthroughput assays of unbound oligomers or oligomers bound to a substrate(such as, for example, an oligonucleotide microarray).

[0186] The substrate from which the antibody microarray is formed may becomprised of any suitable material, including, for example, silicon,aluminum, glass, plastic, polycarbonate, polystyrene, polypropylene,polyethylene, agarose beads, or membranes made of nitrocellulose, nylon,or polyvinylidene difluoride (PVDF). The substrate is typically in theform of a “chip” having a flat surface, but may also comprise otheralternative configurations such as, for example, beads, membranes,microparticles, and the interior surface of reaction vessels.

[0187] Suitable methods of immobilizing (i.e., attaching directly orindirectly by covalent bonds, non-covalent bonds, or other means)antibodies on suitable substrates are known to those skilled in the art.The antibody microarrays may have any desirable density (i.e., spots ofantibodies/area), including a high-density of antibodies. Antibodies maybe directly immobilized on a substrate, for instance, by covalentlyattaching the antibodies to a glass microscope slide. Antibodies may beindirectly immobilized on a substrate, for example, by immobilizingprotein A or G on the substrate and then immobilizing the antibodies onthe substrate through the antibodies' interactions with protein A or G.Another example of a suitable method comprises immobilizing an antibodywithin a three-dimensional hydrophilic polymer matrix on a glassmicroscope slide. A coupling reagent in the matrix may react with theantibody to covalently attach and immobilize the antibody within thematrix. Yet another suitable method for immobilizing antibodies on asubstrate involves printing or spotting the antibodies onto a substrateby jet droplet printing methods such as, for example, the methoddescribed in U.S. Pat. No. 5,958,342, incorporated herein by referencein its entirety.

[0188] The following references, each of which is incorporated herein byreference in its entirety, also describe examples of suitable methodsfor immobilizing antibodies on suitable substrates: MacBeath andSchreiber, “Printing Proteins as Microarrays For High-ThroughputFunction Determination,” Science 289: 1760, 8 September 2000; Haab, B.B.; Dunham, M. J.; Brown, P. O., “Protein microarrays for highlyparallel detection and quantitation of specific proteins and antibodiesin complex solutions,” Genome Biology, 2001, 2(2):research0004.1-0004.12; and U.S. Pat. No. 6,197,599. In addition, othermethods known to those skilled in the art may also be used to immobilizethe antibodies described herein to a suitable substrate to form anantibody microarray in accordance with the present invention.

[0189] The antibodies may be immobilized at predetermined positions onthe substrate so that, if desired, the antibodies can be identified bytheir specific position. Once immobilized, the antibodies retain theirbinding capability and specificity, and the variable regions of theantibodies will be fully exposed to interact with antigens (such asoligonucleotides having one or more organic protecting groups covalentlybound thereto). Separate discrete regions (e.g., spots or othergeometric or non-geometric shapes) of the antibody chip may includeantibodies with the same or different binding specificity. In such anembodiment, each discrete region is preferably separated by areas notcontaining antibodies. Typically, each discrete region will only haveantibodies with the same binding specificity in order to determine whichprotecting group or groups was not deprotected by detecting the presenceof binding in each discrete region.

[0190] The antibodies on the antibody microarrays of the presentinvention may also be integrated into flow channels, flow chambers, orwells on or in the substrate so that the antibodies are always inaqueous solution or other suitable solution, thus ensuring theantibodies will not be denatured.

[0191] An antibody microarray according to the present invention may beused to detect incomplete deprotection of synthetic oligonucleotidesand/or separate protected from deprotected synthetic oligonucleotides.An antibody microarray according to the present invention may also beused to detect incomplete elongation of synthetic oligonucleotidesand/or separate full-length from incomplete synthetic oligonucleotides.Various embodiments are possible for such uses of an antibodymicroarray.

[0192] For example, free oligonucleotides that are synthesized for useon an oligonucleotide microarray may be contacted with an antibody chipthat contains an array of antibodies designed to bind tooligonucleotides having some or all of the protecting groups used in thesynthesis of the oligonucleotides. That is, the antibody microarraycontains antibodies specific for at least one, preferably more than one,more preferably all of the protecting groups used in the synthesis ofthe oligonucleotides. The oligonucleotides that are not fullydeprotected will be bound to one of the antibodies having bindingspecificity to the protecting group remaining on the oligonucleotide.Such a method would increase the purity of the unbound syntheticoligonucleotides that are to be immobilized (e.g., by printing,spotting, or other known methods) on the substrate of an oligonucleotidearray. Using an antibody microarray having antibodies specific for allthe protecting groups employed in the synthesis of the syntheticoligonucleotides, the method could be used to ensure that theoligonucleotides are fully deprotected and are full-length before beingadded to an oligonucleotide array. The antibodies of such an embodimentcould be immobilized on a surface (flat or otherwise) of the substrateor could be immobilized in flow channels or flow chambers that thesynthetic oligonucleotides could be passed through. Such flow channelsor chambers may be used to ensure that the antibodies would remain in anappropriate solution or buffer to prevent denaturation and may possiblyincrease the throughput of detection or separation methods. Such anantibody chip could also be used to determine the specific protectinggroups that were present in the synthetic oligonucleotide oroligonucleotides by correlating known locations of the specificantibodies on the antibody microarray with the detection of the presenceof binding at specific locations.

[0193] It may also be possible to contact an antibody chip of thepresent invention with a fully or partially complete oligonucleotidemicroarray in order to detect insufficient deprotection and/orelongation of the oligonucleotides thereon. The antibody microarray maybe formulated such that each oligonucleotide in the oligonucleotidearray is subjected to the appropriate antibody or antibodies. Aftercontacting the antibody chip with the fully or partially completeoligonucleotide microarray, a suitable detection method could be used todetermine the location and identity of any protecting groups remainingon the oligonucleotide microarray. As described above, an indicia of thelocation of any incomplete deprotection/elongation could also begenerated.

[0194] One aspect of the invention includes a method of detectingincomplete deprotection of a synthetic test oligonucleotide using anantibody microarray. The method comprises the following steps:

[0195] (a) providing an antibody microarray comprising a plurality ofantibodies immobilized on a substrate, each antibody specificallybinding to a synthetic oligonucleotide having an organic protectinggroup covalently bound thereto, which antibody does not bind to thesynthetic oligonucleotide when the organic protecting group is notcovalently bound thereto;

[0196] (b) contacting the synthetic test oligonucleotide with theantibodies immobilized on the antibody microarray; and

[0197] (c) detecting binding of the synthetic test oligonucleotide tothe antibodies immobilized on the antibody microarray, the presence ofbinding indicating incomplete deprotection of the synthetic testoligonucleotide. Such a method may optionally include a separation stepbefore step (c) in order to remove any unbound syntheticoligonucleotides from the antibody microarray, thus avoiding thedetection of the unbound synthetic oligonucleotides in step (c).

[0198] Another aspect of the invention includes a method of purifying asynthetic oligonucleotide mixture prepared using organic protectinggroups. The method uses an antibody microarray and comprises thefollowing steps:

[0199] (a) providing a synthetic oligonucleotide mixture comprisingfully deprotected synthetic oligonucleotides, partially deprotectedsynthetic oligonucleotides, and/or fully protected syntheticoligonucleotides, the mixture having at least one partially or fullyprotected synthetic oligonucleotide with a protecting group covalentlybound thereto;

[0200] (b) providing an antibody microarray comprising a plurality ofantibodies immobilized on a substrate, each antibody specificallybinding to a synthetic oligonucleotide having an organic protectinggroup covalently bound thereto, which antibody does not bind to thesynthetic oligonucleotide when the organic protecting group is notcovalently bound thereto, the plurality of antibodies including at leastone antibody that is capable of binding at least one of the partially orfully protected synthetic oligonucleotides;

[0201] (c) contacting the synthetic oligonucleotide mixture with theantibodies immobilized on the antibody microarray such that at least oneof the protected synthetic oligonucleotides from the mixture is bound toat least one of the antibodies immobilized on the antibody microarray,wherein at least a portion of the oligonucleotides in the syntheticoligonucleotide mixture do not bind to the plurality of antibodies; and

[0202] (d) separating the antibody microarray and the unbound syntheticoligonucleotides, thus separating the unbound synthetic oligonucleotidesfrom any partially and/or fully protected synthetic oligonucleotide oroligonucleotides bound to the antibodies immobilized on the antibodymicroarray.

[0203] Yet another aspect of the present invention includes a method ofpreparing an oligonucleotide microarray using an antibody microarray.The method comprises the following steps:

[0204] (a) providing a synthetic oligonucleotide mixture prepared usingorganic protecting groups, the mixture comprising fully deprotectedsynthetic oligonucleotides, partially deprotected syntheticoligonucleotides, and/or fully protected synthetic oligonucleotides, themixture having at least one partially or fully protected syntheticoligonucleotide with a protecting group covalently bound thereto;

[0205] (b) providing an antibody microarray comprising a plurality ofantibodies immobilized on a first substrate, each antibody specificallybinding to a synthetic oligonucleotide having an organic protectinggroup covalently bound thereto, which antibody does not bind to thesynthetic oligonucleotide when the organic protecting group is notcovalently bound thereto, the plurality of antibodies including at leastone antibody that is capable of binding at least one of the partially orfully protected synthetic oligonucleotides;

[0206] (c) contacting the synthetic oligonucleotide mixture with theantibodies immobilized on the antibody microarray such that at least oneof the protected synthetic oligonucleotides from the mixture is bound toat least one of the antibodies immobilized on the antibody microarray,wherein at least a portion of the oligonucleotides in the syntheticoligonucleotide mixture do not bind to the plurality of antibodies;

[0207] (d) separating the antibody microarray and the unbound syntheticoligonucleotides, thus separating the unbound synthetic oligonucleotidesfrom any partially and/or fully protected synthetic oligonucleotide oroligonucleotides bound to the antibodies immobilized on the antibodymicroarray; and

[0208] (e) immobilizing the unbound synthetic oligonucleotides on asecond substrate to form an oligonucleotide microarray.

[0209] In any of the above aspects of the invention, the plurality ofantibodies immobilized on the substrate may include one or more (e.g.,at least two) sets of antibodies, with each set of antibodies having adifferent binding specificity from the other sets. In some embodiments,each set of antibodies is localized to a separate discrete region on thesubstrate, with each discrete region being separated by a region on thesubstrate to which no antibodies are immobilized. In addition, theantibodies of the antibody microarray may be immobilized in flowchannels or flow chambers formed in or on the substrate of the antibodymicroarray.

[0210] As discussed above, each antibody immobilized on the antibodymicroarray specifically binds to a synthetic oligonucleotide having anorganic protecting group covalently bound thereto, which antibody doesnot bind to the synthetic oligonucleotide when the organic protectinggroup is not covalently bound thereto. It is again noted that antibodiesof the invention may be characterized by their selective binding toparticular “reagent” or “benchmark” oligonucleotides, but the sameantibodies may also bind to a variety of other oligonucleotides (e.g.,longer or shorter nucleotides) or other compounds that contain the sameprotecting group. In some embodiments, each antibody may becharacterized in that the synthetic oligonucleotide to which theantibody specifically binds includes a protected nucleotide according toFormula (I)

[0211] wherein:

[0212] R is H or a protecting group; subject to the proviso that R is acovalent bond to an adjacent nucleotide when the protected nucleotide isnot a 5′ terminal nucleotide in the oligonucleotide;

[0213] R¹ is H or a protecting group; subject to the proviso that R¹ isa covalent bond to an adjacent nucleotide when the protected nucleotideis not a 3′ terminal nucleotide in the oligonucleotide;

[0214] R² is H or —OR³;

[0215] R³ is H or a protecting group;

[0216] Base is a purine or pyrimidine base;

[0217] R⁴ is a protecting group substituted for a hydrogen on an aminogroup of the base, subject to the proviso that R⁴ is not present inFormula (I) when R, R¹, or R³ is a protecting group or when the basedoes not include an amino group; and

[0218] further subject to the proviso that only one of R, R¹, R³, and R⁴is a protecting group and the others of R, R¹, R³, and R⁴ are notprotecting groups.

[0219] In particular embodiments, R is a protecting group comprisingdimethoxytrityl; R¹ is a protecting group comprising β-cyanoethyl; R² is—OR³ and R³ is a protecting group comprising tert-butyldimethylsilyl;and R⁴ is a protecting group selected from the group consisting ofacetyl, benzoyl, dimethylformamidine, isobutyrl, phenoxyacetyl, andisopropyl-phenoxyacetyl. In other particular embodiments, theoligonucleotide to which the antibody selectively binds consists of from3 to 20 nucleotides.

[0220] The synthetic oligonucleotides (or synthetic testoligonucleotides) used in the above methods may include a detectablemarker. Such a detectable marker may be a radio label, a member of abinding pair such as biotin or avidin, a fluorophore such as greenfluorescent protein, a detectable enzyme, and/or a dye. Other possibleexamples of detectable markers include rhodamine, fluorescein, digoxin,Cy3 dye, Cy5 dye, and dinitrophenol (DNP). In addition, any otherdetectable marker suitable for the specific method could also be used.In the method of detecting incomplete deprotection of a synthetic testoligonucleotide (or any of the other methods where detection of bindingis desirable), the binding of the synthetic test oligonucleotide to theantibodies immobilized on the antibody microarray may be detected bydetecting the detectable marker of the synthetic test oligonucleotide.

[0221] 11. Pre-Impregnation of Oligonucleotide Microarrays withAntibodies.

[0222] The present invention also includes an oligonucleotide arrayimpregnated with antibodies. Each of the antibodies specifically bindsto a synthetic oligomer having an organic protecting group covalentlybound thereto, as described above. As used herein, “impregnated” meansthat antibodies are immobilized on an oligonucleotide array and“pre-impregnation” means that antibodies are immobilized of on asubstrate that will eventually include an array of oligonucleotides.

[0223] According to this aspect of the present invention, the antibodiesare pre-impregnated (i.e., immobilized) on a substrate that willeventually contain an oligonucleotide microarray. The antibodies may beimmobilized on a substrate by any method known in the art, such as, forexample, the methods described above in connection with antibody chipsof the present invention.

[0224] After the antibodies have been immobilized on the substrate, theoligonucleotide array is formed on the microarray, either by synthesisin situ on the substrate or by polymerizing the oligonucleotidesseparately and then linking them to the appropriate regions of thesubstrate.

[0225] In some applications, it may be necessary to encapsulate theantibodies or block the binding sites on the antibodies with one or moreblocking groups until such time as detection of incomplete deprotectionand/or insufficient elongation is desired. In such embodiments, theantibodies may be contacted with the oligonucleotides when desired byreleasing the antibodies from the capsules or removing the blockinggroups from the antibodies. A suitable assay may then be performed todetermine incomplete deprotection and/or elongation.

[0226] The present invention is explained in greater detail in thefollowing non-limiting examples.

EXAMPLE 1 Synthesis of Olionucleotides

[0227] Synthesis was performed on an ABI DNA/RNA Synthesizer, Model 394(PE Biosystems, 850 Lincoln Centre Drive, Foster City, Calif. 94404)according to manufacturer's protocol. Slightly modified 1 micromolarscale cycle was used during synthesis (see manufacturer's instructions).The primary starting materials (and suppliers/manufacturers inparentheses) were as follows:

[0228] Activator (0.45 M tetrazole in acetonitrile), CAP A (aceticanhydride/tetrahydrofuran/2,6 lutidine), CAP B (N-methylimidazole/tetrahydrofuran) and oxidizer (0.02 M iodine/pyridine/THF/H2O)(Prime Synthesis)

[0229] Pac-dA(5′-dimethoxytrityl-N-phenoxyacetyl-2′-deoxyAdenosine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Glen Research)

[0230] Ipr-Pac-dG(5′-dimethoxytrityl-N-p-isopropyl-phenoxyacetyl-2′-Guanosine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Glen Research)

[0231] Ac-dC(5′-dimethoxytrityl-acettyl-2′-deoxycytidine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Glen Research)

[0232] dmf-G(5′-dimethoxytrityl-dimethylformamidine-Guanosine,2′-O-TBDMS-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Glen Research)

[0233] Bz-dC---CPG beads (5′-dimethoxytrityl-N-benzoyl-2′-deoxycytidine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite-succinyllinker-beads (3000 Ang) (CPG Inc.)

[0234] Ibu-dG---CPG beads(5′-dimethoxytrityl-N-isobutyl-2′-deoxycytidine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite-succinyllinker-beads (3000 Ang) (CPG Inc.)

[0235] The following compounds were synthesized, the compounds beinglinked to beads as shown:

[0236] Pac-dA---Pac-dA---Bz-dC---succinyl linker---Beads

[0237] Pac-dA---Pac-dA---Ibu-dG---succinyl linker---Beads

[0238] Ipr-Pac-dG---Ipr-Pac-dG---Bz-dC---succinyl linker---Beads

[0239] Ipr-Pac-dG---Ipr-Pac-dG---Ibu-dG---succinyl linker---Beads

[0240] Ac-dC---Ac-dC---Bz-dC---succinyl linker---Beads

[0241] Ac-dC---Ac-dC---Ibu-dG---succinyl linker---Beads

[0242] dmf-G---dmf-G---Bz-dC---succinyl linker---Beads

[0243] dmf-G---dmf-G---Ibu-dG---succinyl linker---Beads

[0244] The foregoing compounds were administered directly to animals asan immunogen, without separating the oligonucleotide from the solidsupport, for the production of antibodies, as further described inExample 2 below.

EXAMPLE 2 Innoculation of Animals

[0245] Female BALB/c mice of eight to twelve weeks old were purchasedfrom Charles River, Raleigh, N.C., USA. The mice were housed in caseswith filter caps.

[0246] After oligonucleotide chain synthesis was completed as describedin Example 1, the beads with nucleotides were gently crushed byhand-pressuring the glass plates, between which beads were positioned.

[0247] 5 μM of each eight oligonucleotides mentioned above were mixed in4 ml PBS (150 mM sodium chloride in 100 mM phosphate buffer, pH 7.2).

[0248] The mixture was thoroughly vortexed suspending the crushed beads.150 μL of the vortexed mixture was taken and added into 300 μL of PBS ina syringe. Just before injection, the solution containing beads wasmixed again by shaking the syringe to suspend the broken beads. Then 150μL or 300 μL of well-mixed solution was injected into mouse peritonealcavity. This procedure was used for the first injection and thefollowing boosts. Injection time schedule: Injection Date (day) first  0second  14th third  28th  4th  42nd  5th  56th  6th  70th  7th  84th 8th  98th  9th 112th 10th 138th 11th (final, 4 day before fusion) 142nd

[0249] Four days after the final injection, spleen cells are harvestedfrom the animals and fused with myeloma cells (P3x.63.Ag8.653) inaccordance with known techniques to produce hybridoma cell lines, whichare then screened to determine the binding characteristics as describedbelow to isolate particular cell lines that produce the desired antibodyof the invention.

EXAMPLE 3 Immunodot-Blot Assay for Antibody Characterization

[0250] The Immunodot-blot assay involves UV cross linking ofoligonucleotides onto membrane paper, and is directly applicable to atest kit for detection, identification and quantifying the protectinggroups on product oligomers. This procedure may be carried out asfollows: (a) wet membrane paper with TBS (10 mM Tris, pH 7.2; 150 mMNaCl); (b) blot oligonucleotides to be tested onto membrane paper undervacuum; (c) UV cross link nucleotide onto membrane paper; (d) blockmembrane paper with 1% casein-TBST (TBS plus Tween 20, 0.1% by volume)at room temperature for 2 hr or 4° C. overnight; (e) wash membrane withTBST 3 times, each for 15 min; (f) form antigen-antibody complex byincubation of plate with sample be tested (diluted in 1% casein-TBST) atroom temperature for 1 hr; (g) wash as above; (h) react with secondantibody conjugate (diluted in 1% casein-TBST) at room temperature for 1hr; (i) wash as above; (j) develop color reaction by incubation ofmembrane with substrate solution.

EXAMPLE 4 Dot-Blot Assay of Monoclonal Antibody 1H11

[0251] Monoclonal antibody 1 H11, produced as described in Example 2above, was characterized by a dot-blot assay as described in Example 3above. Results are shown as a bar graph in FIG. 1. In FIG. 1, lanes (orcolumns) 1 and 2 represent oligoPac-dA20mers treated with NH₄OH for 6hours at 65° C. and 15 minutes at 4° C., respectively. Columns 3 and 4represent oligoBz-dC20mers treated with NH₄OH for 6 hours at 65° C. and15 minutes at 4° C., respectively. Columns 5 and 6 representoligoAc-dC20mers treated with NH₄OH for 6 hours at 65° C. and 15 minutesat 4° C., respectively. Columns 7 and 8 represent oligoIpr-Pac-dG20merstreated with NH₄OH for 6 hours at 65° C. and 15 minutes at 4° C.,respectively. Columns 9 and 10 represent oligoIbu-dG20mers treated withNH₄OH for 6 hours at 65° C. and 15 minutes at 4° C., respectively.Columns 11, 12 and 13 represent oligodT20mers, completely deprotected,with DMT group only, and with cyanoethyl group only, respectively.Antibody activity is given as optical density (479 nm) from ELISA(Example 7 below), and the positive or negative result of the dot blotassay is given in the open or filled circle appearing over each columnin the bar graph. Note the activity of monoclonal antibody 1 H11 inselectively binding to the oligoIbu-dG20mer in column 10.

EXAMPLE 5 Dot-Blot Assay of Monoclonal Antibody 7H3

[0252] Monoclonal antibody 7H3, produced as described in Example 2above, was characterized by a dot-blot assay as described in Example 3above. Results are shown as a bar graph in FIG. 2. In FIG. 2, lanes (orcolumns) 1 and 2 represent oligoPac-dA20mers treated with NH₄OH for 6hours at 65° C. and 15 minutes at 4° C., respectively. Columns 3 and 4represent oligoBz-dC20mers treated with NH₄OH for 6 hours at 65° C. and15 minutes at 4° C., respectively. Columns 5 and 6 representoligoAc-dC20mers treated with NH₄OH for 6 hours at 65° C. and 15 minutesat 4° C., respectively. Columns 7 and 8 represent oligoIpr-Pac-dG20merstreated with NH₄OH for 6 hours at 65° C. and 15 minutes at 4° C.,respectively. Columns 9 and 10 represent oligoIbu-dC20mers treated withNH₄OH for 6 hours at 65° C. and 15 minutes at 4° C., respectively.Columns 11, 12 and 13 represent oligodT20mers, completely deprotected,with DMT group only, and with cyanoethyl group only, respectively.Antibody activity is given as optical density as described above, andthe positive or negative result of the dot blot assay is given in theopen or filled circle appearing over each column in the bar graph. Notethe activity of monoclonal antibody 7H3 in selectively binding to theoligoBz-dC20mer in column 4.

EXAMPLE 6 Western Blot Assay for Antibody Characterization

[0253] The Western blot assay involves low voltage transfer ofoligonucleotides from gel to membrane paper and UV cross linking ofoligonucleotides onto the membrane. This assay may be carried out asfollows: (a) cast 15% non-denaturing gel containing 10 mM MgCl; (b) loadoligonucleotides (oligomers) into the wells of the gel; (c) run gel at200 voltage in ice bath; (d) transfer oligonucleotides from gel tomembrane paper at 25 voltage for 25 min in ice bath; (e) UV cross linkpolynucleotides on membrane; (f) block membrane paper with 1%casein-TBST at room temperature for 2 hr or 4° C. overnight; (g) washmembrane with TBST 3 times, each for 15 min; (h) incubate samples betested (diluted in 1% casein-TBST) at room temperature for 1 hr; (i)wash as above; (j) incubate membrane with second antibody conjugate(diluted in 1% casein-TBST) at room temperature for 1 hr; (k) wash asabove; and (l) color-develop by incubation of membrane with substratesolution.

EXAMPLE 7 Detection of Antibody Using Biotinylated Polynucleotides asAntigen and an ELISA involving Streptavidin-Biotin System

[0254] An enzyme-linked immunosorbent assay (ELISA) for the detection ofthe antibody is carried out as follows: (a) pre-screen microtiter platethat is pre-coated with streptavidin; (b) coat the plate with apreparation of biotinylated oligonucleotide or other materials to betested (at 5 μg/ml in PBS)(PBS: 150 mM NaCl, 10 mM Phosphate buffer, pH7.4), then incubate at room temperature for 2 hrs; (c) wash 3 times with0.1% Tween in PBS (PBST), each for 15 min; (d) block with 1% casein inPBST at room temperature for hrs or 4° C. overnight; (e) wash as above;69 form antigen-antibody complex by incubation of plate with antibody(or antibodies) at room temperature for 1 hr; (g) wash as above; (h)react with second antibody-peroxidase conjugate (in 1% casein-PBST) atroom temperature for 1 hr; (i) wash as above; (j) develop color reactionby adding tetramethylbenzidine (TMB) solution (TMB solution: 42 mM TMB,0.004% H₂O₂, 0.1 M acetate buffer, pH 5.6) and incubating at roomtemperature for 15 min, then stop the reaction with 2 M H₂SO₄; and (k)read absorption value at 469 nm.

EXAMPLE 8 ELISA and Dot-Blot Assay of Monoclonal Antibody AgainstBenzoyl, Isobutryl, and Isopropyl-Phenoxyacetyl

[0255] Monoclonal antibodies (mAb) against protecting groups benzoyl(Bz), isobutryl (ibu), and isopropyl-phenoxyacetyl (ipr-Pac), producedas described in Example 2 above, were characterized by a standard ELISAassay and a dot-blot assay as described in Example 3 above. An ELISAassay developed with biotinylated nucleic acids of 20 residues eachattached to a 96-well microtiter plate demonstrated the specificity ofthe antibodies for their respective antigens. FIG. 3A, FIG. 4A, and FIG.5A show results for monoclonal antibodies against Bz, ibu, and ipr-Pac,respectively. The figures show completely deprotected (<1% Bz remaining)homopolymers of dC residues, designated oligo dC(Bz), ie. originallyprotected with Bz (lane 1, open bar), protected (>97% Bz remaining)oligo Bz-dC (lane 2, shaded bar), completely (<1% ipr-Pac remaining)deprotected oligo dG(ipr-Pac) (lane 3), protected (>76% ipr-Pac) oligoipr-PacdG (lane 4), completely (<1% ibu remaining) deprotected oligodG(ibu) (lane 5), protected (>91% ibu remaining) oligo ibu-dG (lane 6),and completely deprotected oligo dT (lane 7). The dT polymer had but oneprotecting group, dimethyltrityl (DMT) that was removed from the 5′OH ofthe 5′-terminal residue with mild acid. Finally, lane 8 shows oligo dTwith DMT remaining.

[0256] Dot-Blot assays of anti-Bz mAb, anti-ibu mAb, and anti-ipr-PacmAb activities were performed in which the 20mer DNAs were linked tonitrocellulose membrane by UV. The amounts of 20mer DNA applied to themembrane are shown on the left of FIG. 3B, FIG. 4B, and FIG. 5B anddemonstrate the level of sensitivity of the assay. The DNAs used to testanti-Bz mAb were those described for the ELISA plus deprotected oligodA(Bz), protected oligo Bz-dA, oligo dC(ibu), oligo ibu-dC, oligodA(ibu) and oligo ibu-dA. FIG. 3B shows that the anti-Bz mAb recognizedthe protecting group on dA and dC. The DNAs used to test anti-ibu mABwere those described for the ELISA plus protected oligo ibu-dA,deprotected oligo dA(ibu), oligo ibu-dC, oligo dC(ibu) and all are notedat the top of the dot-blot. FIG. 4B shows that the anti-ibu mAbrecognized ibu on dG, the most common use of the protecting group, butalso on dA. The DNAs used to test anti-ipr-Pac mAb were those describedfor the ELISA plus protected oligo ibu-dA, deprotected oligo dA(ibu),oligo ibu-dC, oligo dC(ibu), oligo Bz-dA, oligo dA(Bz) and all are notedat the top of the dot-blot. FIG. 5B shows that the anti-ipr-Pac mAbrecognized ipr-Pac on dG, the most common use of the protecting group,but also on dA and dC. The mAb also recognized the ibu protecting group(ibu-dG, ibu-dA and ibu-dC). This cross-reactivity indicates that theantibody was highly selective in its identification of a chemistrycommon to both ipr-Pac and ibu, possibly CH(CH₃)₂. Thus the anti-ibu andanti-iprPac mAbs could be used in combination to identify the protectinggroup remaining on an oligo.

[0257] Greater amounts of DNA were tested in a dot blot assay ofanti-ibu mAb (FIG. 4C). The results of this experiment demonstrated thatthe ibu protecting group was recognized by the mAb no matter whichnucleobase was protected.

[0258]FIG. 3C, FIG. 4D, and FIG. 5C demonstrate that partiallydeprotected oligomers can be re-treated to remove the remainingprotecting groups, and re-tested with mAb. FIG. 3C shows that anti-BzmAb recognized re-deprotected oligomer oligo Bz-dC (center column).Likewise, FIG. 4D shows that anti-ibu mAb recognized re-deprotectedoligomer oligo ibu-dG (center column) and FIG. 5C shows thatanti-ipr-Pac mAb recognized re-deprotected oligomers oligo ipr-Pac-dGand oligo ibu-dG (columns second from left and forth from left,respectively). Thus, this approach is applicable to quality controlwithout having to discard expensive nucleic acid samples.

[0259] An RNA standard with protecting groups Bz, ibu and ipr-Pac wassynthesized and assayed for identification of the protecting groups withthe mAb against Bz (FIG. 3D), ibu (FIG. 4E), and ipr-Pac (FIG. 5D).Dot-blot assays clearly show that the monoclonal antibodies do notdifferentiate RNA from DNA. Although there was a higher backgroundsignal with RNA than with DNA, there was a significant distinctionbetween RNA with and without protecting groups, especially at the loweramounts of RNA. The amount of RNA on the membrane was estimated from theoptical absorbance of the sample.

EXAMPLE 9 mAb Dot-Blot Assay of Protecting Groups Vs HPLC

[0260] Dot-blot detection of Bz groups remaining on a standardized 20meroligo dC molecule were performed as described in Example 3. Completelydeprotected and the untreated oligo dC 20mers were analyzed for the Bzprotecting group using a totally independent and differentquantification method. The two oligomers were hydrolyzed to theconstituent nucleosides and then their nucleoside composition identifiedand quantified using a recognized high performance liquid chromatography(HPLC) method with concentrated samples. Because of the lack ofsensitivity, HPLC detection required 50-100 fold the amounts of Bz-dCused in the mAb assays (see FIG. 7). FIG. 6A shows the result of anti-BzmAb tested against nmole amounts of Bz groups on protected oligo Bz-dC(right column) and the same nmole amounts of Bz- on Bz-dC (left column).Each amount of Bz-dC oligo was diluted with completely deprotected dColigo of the same length (20mer) to demonstrate the sensitivity of themAb detection even in the presence of 2500-fold dC (ie. 0.04%). The mAbassay demonstrated that the mAb could detect the Bz group on DNA even inthe presence of a 2500-fold excess of dC in DNA.

[0261] The dot-blot shown in FIG. 6A was subjected to densitometry toquantitate the mAb response. After background subtraction, the remainingdensity was plotted as a function of Bz groups in oligo Bz-dC determinedby HPLC (FIG. 6B). The data indicated that the high sensitivity of theanti Bz mAb detection was linear in 0.1-1.0 nmol range.

[0262] Next, it was determined whether the mAb response could beenhanced with an increase in the amount of DNA on the dot-blot membrane.The amount of Bz was determined by standard HPLC methods. Thisexperiment showed that detection of the Bz protecting group in a mixtureof the protected sample with the deprotected sample at a ratio of 1/2500could be enhanced by increasing the amount of DNA on the membrane,though the ratio was maintained (FIG. 6C).

[0263] Finally, experiments were conducted to show a direct comparisonof the mAb and HPLC detection of Bz. Anti-Bz mAb was utilized in adot-blot assay to detect Bz on dC in the oligo Bz-dC (20mer). Thedensity response of the Bz group detected Bz by the mAb assay andquantified by densitometry was plotted against the amount of Bz in theDNA on each dot (FIG. 7A). The amount of Bz in the DNA was calibrated bydigestion of a large amount of DNA and analysis by HPLC identificationand quantification of the Bz-dC mononucleoside. For HPLC experiments,three samples of Bz-dC oligo were hydrolyzed and analyzed forcomposition by HPLC. The response of the UV-diode array detector wasplotted against the amount of Bz in the samples (FIG. 7B). The sampleamounts were determined by comparison to samples “spiked” with knownamounts of Bz-dC. The amounts of Bz-dC added to samples as spikes werefrom a weighed stock of Bz-dC. Thus, the HPLC response was calibratedwith known amounts of Bz-dC. The results of these experiments show thatthe detection of Bz by anti-Bz mAb was within the pmole range whereasHPLC detection of Bz was limited to the nmole range.

EXAMPLE 10 Detection of Remaining Protecting Groups in CommercialSamples

[0264] A blind study was conducted to demonstrate the detection ofremaining protecting groups in commercial samples by mAb. The purpose ofthe this experiment was to determine if protecting groups could bedetected and identified with mAb technology in presumably completelydeprotected samples that had been treated as commonly accomplished inthe oligo synthesis industry. The nature of the protecting groups usedby eight selected companies was not known, thus the experiment was ablind study. Two 20mer oligos (oligo dA-dC and oligo dG-dT) from each ofthe eight companies were ordered to be synthesized and deprotected, andsalt removed under as identical conditions as possible. The oligos wereshipped by express mail, as is often the case, and then subjected to mAbanalysis by dot blot. The dA-dC oligo from one company (#6), andpossibly a second (#2), had remaining Bz protecting groups as determinedby anti-Bz mAb testing (FIG. 8A). The dG-dT oligos from two companies(#2 and #6) had ipr-Pac protecting groups remaining as determined byanti-ipr-Pac mAb (FIG. 8B). The remaining protecting groups in thecommercial samples were confirmed by increasing amounts of sample andfurther deprotection and re-analyses. The oligo dA-dC samples fromcompanies #2 and #6 were tested in higher amounts to confirm thepresence of the Bz protecting group. In addition, the samples weretreated to remove the remaining protecting groups using a standardprotocol. The re-analysis after further deprotection indicated that thegroups were now removed (FIG. 8C). This also demonstrates that expensivenucleic acid samples can be re-treated to remove protecting groups andthat they need not be discarded. The oligo dG-dT samples were re-treatedto remove remaining protecting groups and re-analyzed with anti-ipr-PacmAb with the result that the ipr-Pac group could be removed withoutsacrificing the DNA (FIG. 8D).

EXAMPLE 11 Polyclonal Antibody Against Dimethyltrityl

[0265] Production and analyses of polycolonal antibody against the 5′terminal protecting group, dimethyltrityl (DMT) were as described inExample 2. Four mice were inoculated with DMT and sera were drawn fromthe mice after some weeks of boosting with antigen. DMT [DMT-OH], threeDMT at the 5′-end of the deoxynucleotide trimer d(T)₃ [(DMT)₃-d(T)₃],three DMT at the 5′-end of the deoxynucleotide 20mer d(T)₃ with3′-biotin [(DMT)₃-d(T)₂₀-biotin], one DMT at the 5′-end of thedeoxynucleotide 20mer d(T)₂₀ with 3′-biotin [DMT-d(T)₂₀-biotin], the dT20mer with 3′-biotin [d(T)₂₀-biotin], one DMT with biotin [DMT-biotin]and tris-borate saline control were applied to a nitrocellulose membranethat was then assayed with mouse sera (inoculated mice #1-4 and acontrol serum, normal) to assess anti-DMT antibody, mild acid to revealpresence of the DMT (TBS), and avidin to reveal the presence of biotin(FIG. 9). Sera from mice #2 and #4 recognized DMT [as (DMT)₃-d(T)₃],whereas mice #1, #3, and the normal mouse did not. Mild acid revealedthe presence of DMT as a yellow color (not visible in figure) and avidinrevealed the presence of biotin.

[0266] The foregoing is illustrative of the present invention, and isnot to be construed as limiting thereof. The invention is defined by thefollowing claims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A method of detecting incompletedeprotection of a synthetic test oligonucleotide, the method comprisingthe steps of: (a) providing an antibody microarray comprising aplurality of antibodies immobilized on a substrate, each antibodyspecifically binding to a synthetic oligonucleotide having an organicprotecting group covalently bound thereto, which antibody does not bindto the synthetic oligonucleotide when the organic protecting group isnot covalently bound thereto; (b) contacting the synthetic testoligonucleotide with the antibodies immobilized on the antibodymicroarray; (c) detecting binding of the synthetic test oligonucleotideto the antibodies immobilized on the antibody microarray, the presenceof binding indicating incomplete deprotection of the synthetic testoligonucleotide.
 2. The method of claim 1, wherein the plurality ofantibodies immobilized on the substrate includes at least two sets ofantibodies, each set of antibodies having a different bindingspecificity from the other sets.
 3. The method of claim 2, wherein eachset of antibodies is localized to a separate discrete region on thesubstrate, each discrete region being separated by a region on thesubstrate to which no antibodies are immobilized.
 4. The method of claim1, wherein the synthetic oligonucleotide to which the antibodiesspecifically bind includes a protected nucleotide according to Formula(I)

wherein: R is H or a protecting group; subject to the proviso that R isa covalent bond to an adjacent nucleotide when the protected nucleotideis not a 5′ terminal nucleotide in the oligonucleotide; R¹ is H or aprotecting group; subject to the proviso that R¹ is a covalent bond toan adjacent nucleotide when the protected nucleotide is not a 3′terminal nucleotide in the oligonucleotide; R² is H or —OR³; R³ is H ora protecting group; Base is a purine or pyrimidine base; R⁴ is aprotecting group substituted for a hydrogen on an amino group of thebase, subject to the proviso that R⁴ is not present in Formula (I) whenR, R¹, or R³ is a protecting group or when the base does not include anamino group; and further subject to the proviso that only one of R, R¹,R³, and R⁴ is a protecting group and the others of R, R¹, R³, and R⁴ arenot protecting groups.
 5. The method of claim 4, wherein R⁴ is aprotecting group selected from the group consisting of acetyl, benzoyl,dimethylformamidine, isobutyrl, phenoxyacetyl, andisopropyl-phenoxyacetyl.
 6. The method of claim 4, wherein R is aprotecting group comprising dimethoxytrityl.
 7. The method of claim 4,wherein R¹ is a protecting group comprising β-cyanoethyl.
 8. The methodof claim 4, wherein R² is —OR³ and R³ is a protecting group comprisingtert-butyldimethylsilyl.
 9. The method of claim 4, wherein the pluralityof antibodies immobilized on the substrate includes at least two sets ofantibodies, each set of antibodies having a different bindingspecificity from the other sets.
 10. The method of claim 1, wherein theantibodies are immobilized in flow channels or flow chambers formed inor on the substrate of the antibody microarray.
 11. The method of claim1, wherein the synthetic test oligonucleotide includes a detectablemarker.
 12. The method of claim 11, wherein the detectable marker isselected from the group consisting of radio label, biotin, fluorophore,detectable enzyme, and dye.
 13. The method of claim 11, wherein thebinding of the synthetic test oligonucleotide to the antibodiesimmobilized on the antibody microarray is detected by detecting thedetectable marker of the synthetic test oligonucleotide.
 14. The methodof claim 1, wherein: (1) the synthetic oligonucleotide to which theantibodies specifically bind includes a protected nucleotide accordingto Formula (I)

wherein: R is H or a protecting group; subject to the proviso that R isa covalent bond to an adjacent nucleotide when the protected nucleotideis not a 5′ terminal nucleotide in the oligonucleotide; R¹ is H or aprotecting group; subject to the proviso that R¹ is a covalent bond toan adjacent nucleotide when the protected nucleotide is not a 3′terminal nucleotide in the oligonucleotide; R² is H or —OR³; R³ is H ora protecting group; Base is a purine or pyrimidine base; R⁴ is aprotecting group substituted for a hydrogen on an amino group of thebase, subject to the proviso that R⁴ is not present in Formula (I) whenR, R¹, or R³ is a protecting group or when the base does not include anamino group; and further subject to the proviso that only one of R, R¹,R³, and R⁴ is a protecting group and the others of R, R¹, R³, and R⁴ arenot protecting groups; (2) the plurality of antibodies immobilized onthe substrate includes at least two sets of antibodies, each set ofantibodies having a different binding specificity from the other sets;(3) each set of antibodies is localized to a separate discrete region onthe substrate, each discrete region being separated by a region on thesubstrate to which no antibodies are immobilized; (4) the synthetic testoligonucleotide includes a detectable marker selected from the groupconsisting of radio label, biotin, fluorophore, detectable enzyme, anddye; and (5) the binding of the synthetic test oligonucleotide to theantibodies immobilized on the antibody microarray is detected bydetecting the detectable marker of the synthetic test oligonucleotide.15. A method of purifying a synthetic oligonucleotide mixture preparedusing organic protecting groups, the method comprising the steps of: (a)providing a synthetic oligonucleotide mixture comprising fullydeprotected synthetic oligonucleotides, partially deprotected syntheticoligonucleotides, and/or fully protected synthetic oligonucleotides, themixture having at least one partially or fully protected syntheticoligonucleotide with a protecting group covalently bound thereto; (b)providing an antibody microarray comprising a plurality of antibodiesimmobilized on a substrate, each antibody specifically binding to asynthetic oligonucleotide having an organic protecting group covalentlybound thereto, which antibody does not bind to the syntheticoligonucleotide when the organic protecting group is not covalentlybound thereto, the plurality of antibodies including at least oneantibody that is capable of binding at least one of the partially orfully protected synthetic oligonucleotides; (c) contacting the syntheticoligonucleotide mixture with the antibodies immobilized on the antibodymicroarray such that at least one of the protected syntheticoligonucleotides from the mixture is bound to at least one of theantibodies immobilized on the antibody microarray, wherein at least aportion of the oligonucleotides in the synthetic oligonucleotide mixturedo not bind to the plurality of antibodies; and (d) separating theantibody microarray and the unbound synthetic oligonucleotides, thusseparating the unbound synthetic oligonucleotides from any partiallyand/or fully protected synthetic oligonucleotide or oligonucleotidesbound to the antibodies immobilized on the antibody microarray.
 16. Themethod of claim 15, wherein the plurality of antibodies immobilized onthe substrate includes at least two sets of antibodies, each set ofantibodies having a different binding specificity from the other sets.17. The method of claim 15, wherein the synthetic oligonucleotide towhich the antibodies specifically bind includes a protected nucleotideaccording to Formula (I)

wherein: R is H or a protecting group; subject to the proviso that R isa covalent bond to an adjacent nucleotide when the protected nucleotideis not a 5′ terminal nucleotide in the oligonucleotide; R¹ is H or aprotecting group; subject to the proviso that R¹ is a covalent bond toan adjacent nucleotide when the protected nucleotide is not a 3′terminal nucleotide in the oligonucleotide; R² is H or R³; R³ is H or aprotecting group; Base is a purine or pyrimidine base; R⁴ is aprotecting group substituted for a hydrogen on an amino group of thebase, subject to the proviso that R⁴ is not present in Formula (I) whenR, R¹, or R³ is a protecting group or when the base does not include anamino group; and further subject to the proviso that only one of R, R¹,R³, and R⁴ is a protecting group and the others of R, R¹, R³, and R⁴ arenot protecting groups.
 18. The method of claim 17, wherein R⁴ is aprotecting group selected from the group consisting of acetyl, benzoyl,dimethylformamidine, isobutyrl, phenoxyacetyl, andisopropyl-phenoxyacetyl.
 19. The method of claim 17, wherein R is aprotecting group comprising dimethoxytrityl.
 20. The method of claim 17,wherein R¹ is a protecting group comprising β-cyanoethyl.
 21. The methodof claim 17, wherein R² is —OR³ and R³ is a protecting group comprisingtert-butyldimethylsilyl.
 22. The method of claim 17, wherein theplurality of antibodies immobilized on the substrate includes at leasttwo sets of antibodies, each set of antibodies having a differentbinding specificity from the other sets.
 23. The method of claim 15,wherein the antibodies are immobilized in flow channels or flow chambersformed in or on the substrate of the antibody microarray.
 24. The methodof claim 23, wherein step (c) comprises flowing the syntheticoligonucleotide mixture through the flow channels or flow chambers andstep (d) comprises flowing the unbound synthetic oligonucleotides outthe flow channels or flow chambers to separate the unbound syntheticoligonucleotides from the antibody microarray.
 25. The method of claim15, wherein: (1) the synthetic oligonucleotide to which the antibodiesspecifically bind includes a protected nucleotide according to Formula(I)

wherein: R is H or a protecting group; subject to the proviso that R isa covalent bond to an adjacent nucleotide when the protected nucleotideis not a 5′ terminal nucleotide in the oligonucleotide; R¹ is H or aprotecting group; subject to the proviso that R¹ is a covalent bond toan adjacent nucleotide when the protected nucleotide is not a 3′terminal nucleotide in the oligonucleotide; R² is H or —OR³; R³ is H ora protecting group; Base is a purine or pyrimidine base; R⁴ is aprotecting group substituted for a hydrogen on an amino group of thebase, subject to the proviso that R⁴ is not present in Formula (I) whenR, R¹, or R³ is a protecting group or when the base does not include anamino group; and further subject to the proviso that only one of R, R¹,R³, and R⁴ is a protecting group and the others of R, R¹, R³, and R⁴ arenot protecting groups; (2) the plurality of antibodies immobilized onthe substrate includes at least two sets of antibodies, each set ofantibodies having a different binding specificity from the other sets;(3) the antibodies are immobilized in flow channels or flow chambersformed in or on the substrate of the antibody microarray; and (4) step(c) comprises flowing the synthetic oligonucleotide mixture through theflow channels or flow chambers; and (5) step (d) comprises flowing theunbound synthetic oligonucleotides out the flow channels or flowchambers to separate the unbound synthetic oligonucleotides from theantibody microarray.
 26. A method of preparing an oligonucleotidemicroarray comprising the steps of: (a) providing a syntheticoligonucleotide mixture prepared using organic protecting groups, themixture comprising fully deprotected synthetic oligonucleotides,partially deprotected synthetic oligonucleotides, and/or fully protectedsynthetic oligonucleotides, the mixture having at least one partially orfully protected synthetic oligonucleotide with a protecting groupcovalently bound thereto; (b) providing an antibody microarraycomprising a plurality of antibodies immobilized on a first substrate,each antibody specifically binding to a synthetic oligonucleotide havingan organic protecting group covalently bound thereto, which antibodydoes not bind to the synthetic oligonucleotide when the organicprotecting group is not covalently bound thereto, the plurality ofantibodies including at least one antibody that is capable of binding atleast one of the partially or fully protected syntheticoligonucleotides; (c) contacting the synthetic oligonucleotide mixturewith the antibodies immobilized on the antibody microarray such that atleast one of the protected synthetic oligonucleotides from the mixtureis bound to at least one of the antibodies immobilized on the antibodymicroarray, wherein at least a portion of the oligonucleotides in thesynthetic oligonucleotide mixture do not bind to the plurality ofantibodies; (d) separating the antibody microarray and the unboundsynthetic oligonucleotides, thus separating the unbound syntheticoligonucleotides from any partially and/or fully protected syntheticoligonucleotide or oligonucleotides bound to the antibodies immobilizedon the antibody microarray; and (e) immobilizing the unbound syntheticoligonucleotides on a second substrate to form an oligonucleotidemicroarray.
 27. The method of claim 26, wherein the plurality ofantibodies immobilized on the substrate includes at least two sets ofantibodies, each set of antibodies having a different bindingspecificity from the other sets.
 28. The method of claim 26, wherein thesynthetic oligonucleotide to which the antibodies specifically bindincludes a protected nucleotide according to Formula (I)

wherein: R is H or a protecting group; subject to the proviso that R isa covalent bond to an adjacent nucleotide when the protected nucleotideis not a 5′ terminal nucleotide in the oligonucleotide; R¹ is H or aprotecting group; subject to the proviso that R¹ is a covalent bond toan adjacent nucleotide when the protected nucleotide is not a 3′terminal nucleotide in the oligonucleotide; R² is H or —OR³; R³ is H ora protecting group; Base is a purine or pyrimidine base; R⁴ is aprotecting group substituted for a hydrogen on an amino group of thebase, subject to the proviso that R⁴ is not present in Formula (I) whenR, R, or R³ is a protecting group or when the base does not include anamino group; and further subject to the proviso that only one of R, R¹,R³, and R⁴ is a protecting group and the others of R, R, R³, and R⁴ arenot protecting groups.
 29. The method of claim 28, wherein R⁴ is aprotecting group selected from the group consisting of acetyl, benzoyl,dimethylformamidine, isobutyrl, phenoxyacetyl, andisopropyl-phenoxyacetyl.
 30. The method of claim 28, wherein R is aprotecting group comprising dimethoxytrityl.
 31. The method of claim 28,wherein R¹ is a protecting group comprising β-cyanoethyl.
 32. The methodof claim 28, wherein R² is —OR³ and R³ is a protecting group comprisingtert-butyldimethylsilyl.
 33. The method of claim 28, wherein theplurality of antibodies immobilized on the substrate includes at leasttwo sets of antibodies, each set of antibodies having a differentbinding specificity from the other sets.
 34. The method of claim 26,wherein the antibodies are immobilized in flow channels or flow chambersformed in or on the substrate of the antibody microarray.
 35. The methodof claim 34, wherein step (c) comprises flowing the syntheticoligonucleotide mixture through the flow channels or flow chambers andstep (d) comprises flowing the unbound synthetic oligonucleotides outthe flow channels or flow chambers to separate the unbound syntheticoligonucleotides from the antibody microarray.
 36. The method of claim26, wherein: (1) the synthetic oligonucleotide to which the antibodiesspecifically bind includes a protected nucleotide according to Formula(I)

wherein: R is H or a protecting group; subject to the proviso that R isa covalent bond to an adjacent nucleotide when the protected nucleotideis not a 5′ terminal nucleotide in the oligonucleotide; R¹ is H or aprotecting group; subject to the proviso that R¹ is a covalent bond toan adjacent nucleotide when the protected nucleotide is not a 3′terminal nucleotide in the oligonucleotide; R² is H or —OR³; R³ is H ora protecting group; Base is a purine or pyrimidine base; R⁴ is aprotecting group substituted for a hydrogen on an amino group of thebase, subject to the proviso that R⁴ is not present in Formula (I) whenR, R¹, or R³ is a protecting group or when the base does not include anamino group; and further subject to the proviso that only one of R, R¹,R³, and R⁴ is a protecting group and the others of R, R¹, R³, and R⁴ arenot protecting groups; (2) the plurality of antibodies immobilized onthe substrate includes at least two sets of antibodies, each set ofantibodies having a different binding specificity from the other sets;(3) the antibodies are immobilized in flow channels or flow chambersformed in or on the substrate of the antibody microarray; and (4) step(c) comprises flowing the synthetic oligonucleotide mixture through theflow channels or flow chambers; and (5) step (d) comprises flowing theunbound synthetic oligonucleotides out the flow channels or flowchambers to separate the unbound synthetic oligonucleotides from theantibody microarray.
 37. An antibody microarray comprising a pluralityof antibodies immobilized on a substrate, each antibody specificallybinding to a synthetic oligonucleotide having an organic protectinggroup covalently bound thereto, which antibody does not bind to thesynthetic oligonucleotide when the organic protecting group is notcovalently bound thereto.
 38. The antibody microarray of claim 37,wherein the plurality of antibodies immobilized on the substrateincludes at least two sets of antibodies, each set of antibodies havinga different binding specificity from the other sets.
 39. The antibodymicroarray of claim 38, wherein each set of antibodies is localized to aseparate discrete region on the substrate, each discrete region beingseparated by a region on the substrate to which no antibodies areimmobilized.
 40. The antibody microarray of claim 37, wherein thesynthetic oligonucleotide to which the antibodies specifically bindincludes a protected nucleotide according to Formula (I)

wherein: R is H or a protecting group; subject to the proviso that R isa covalent bond to an adjacent nucleotide when the protected nucleotideis not a 5′ terminal nucleotide in the oligonucleotide; R¹ is H or aprotecting group; subject to the proviso that R¹ is a covalent bond toan adjacent nucleotide when the protected nucleotide is not a 3′terminal nucleotide in the oligonucleotide; R² is H or —OR³; R³ is H ora protecting group; Base is a purine or pyrimidine base; R⁴ is aprotecting group substituted for a hydrogen on an amino group of thebase, subject to the proviso that R⁴ is not present in Formula (I) whenR, R¹, or R³ is a protecting group or when the base does not include anamino group; and further subject to the proviso that only one of R, R¹,R³, and R⁴ is a protecting group and the others of R, R¹, R³, and R⁴ arenot protecting groups.
 41. The antibody microarray of claim 40, whereinR⁴ is a protecting group selected from the group consisting of acetyl,benzoyl, dimethylformamidine, isobutyrl, phenoxyacetyl, andisopropyl-phenoxyacetyl.
 42. The antibody microarray of claim 40,wherein R is a protecting group comprising dimethoxytrityl.
 43. Theantibody microarray of claim 40, wherein R¹ is a protecting groupcomprising β-cyanoethyl.
 44. The antibody microarray of claim 40,wherein R² is —OR³ and R³ is a protecting group comprisingtert-butyldimethylsilyl.
 45. The antibody microarray of claim 40,wherein the plurality of antibodies immobilized on the substrateincludes at least two sets of antibodies, each set of antibodies havinga different binding specificity from the other sets.
 46. The antibodymicroarray of claim 37, wherein the antibodies are immobilized in flowchannels or flow chambers formed in or on the substrate of the antibodymicroarray.
 47. The antibody microarray of claim 40, wherein theantibodies are immobilized in flow channels or flow chambers formed inor on the substrate of the antibody microarray.
 48. The antibodymicroarray of claim 45, wherein the antibodies are immobilized in flowchannels or flow chambers formed in or on the substrate of the antibodymicroarray.
 49. The antibody microarray of claim 45, wherein each set ofantibodies is localized to a separate discrete region on the substrate,each discrete region being separated by a region on the substrate towhich no antibodies are immobilized.
 50. An oligonucleotide microarraymade by the method of claim 26.